Phthalazinone derivatives

ABSTRACT

Compounds of the formula (I): 
     
       
         
         
             
             
         
       
     
     wherein A and B together represent an optionally substituted, fused aromatic ring; X can be NR X  or CR X R Y ; if X═NR X  then n is 1 or 2 and if X═CR X R Y  then n is 1; R X  is selected from the group consisting of H, optionally substituted C 1-20  alkyl, C 5-20  aryl, C 3-20  heterocyclyl, amido, thioamido, ester, acyl, and sulfonyl groups; R Y  is selected from H, hydroxy, amino; or R X  and R Y  may together form a spiro-C 3-7  cycloalkyl or heterocyclyl group; R C1  and R C2  are both hydrogen, or when X is CR X R Y , R C1 , R C2 , R X  and R Y , together with the carbon atoms to which they are attached, may form an optionally substituted fused aromatic ring; and
 
R 1  is selected from H and halo.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 15/411,101 filedJan. 20, 2017, which is a continuation of U.S. application Ser. No.14/853,360 filed Sep. 14, 2015, now U.S. Pat. No. 9,566,276, which is acontinuation of U.S. application Ser. No. 14/483,663 filed Sep. 11,2014, now U.S. Pat. No. 9,169,235, which is a continuation of U.S.application Ser. No. 13/179,353 filed Jul. 8, 2011, now U.S. Pat. No.8,912,187, which is a continuation of U.S. application Ser. No.12/109,260 filed Apr. 24, 2008, now U.S. Pat. No. 7,981,889, which is acontinuation of U.S. application Ser. No. 10/876,080 filed Jun. 24,2004, now U.S. Pat. No. 7,449,464, which is a continuation-in-part ofU.S. application Ser. No. 10/799,154 filed Mar. 12, 2004, now abandoned,which claims priority to U.S. Provisional Application Nos. 60/526,244filed Dec. 1, 2003 and 60/454,995 filed Mar. 14, 2003, and furtherclaims foreign priority to United Kingdom Patent Application No.0305681.9 filed Mar. 12, 2003. U.S. application Ser. No. 10/876,080 alsoclaims priority to U.S. Provisional Application No. 60/493,399 filedAug. 6, 2003. All of the above are hereby incorporated by reference intheir entireties.

The present invention relates to phthalazinone derivatives, and theiruse as pharmaceuticals. In particular, the present invention relates tothe use of these compounds to inhibit the activity of the enzyme poly(ADP-ribose)polymerase, also known as poly(ADP-ribose)synthase and polyADP-ribosyltransferase, and commonly referred to as PARP.

The mammalian enzyme PARP (a 113-kDa multidomain protein) has beenimplicated in the signalling of DNA damage through its ability torecognize and rapidly bind to DNA single or double strand breaks(D'Amours, et al., Biochem. J., 342, 249-268 (1999)).

Several observations have led to the conclusion that PARP participatesin a variety of DNA-related functions including gene amplification, celldivision, differentiation, apoptosis, DNA base excision repair and alsoeffects on telomere length and chromosome stability (d'Adda di Fagagna,et al., Nature Gen., 23(1), 76-80 (1999)).

Studies on the mechanism by which PARP modulates DNA repair and otherprocesses has identified its importance in the formation of poly(ADP-ribose) chains within the cellular nucleus (Althaus, F. R. andRichter, C., ADP-Ribosylation of Proteins: Enzymology and BiologicalSignificance, Springer-Verlag, Berlin (1987)). The DNA-bound, activatedPARP utilizes NAD to synthesize poly (ADP-ribose) on a variety ofnuclear target proteins, including topoisomerase, histones and PARPitself (Rhun, et al., Biochem. Biophys. Res. Commun., 245, 1-10 (1998))

Poly (ADP-ribosyl)ation has also been associated with malignanttransformation. For example, PARP activity is higher in the isolatednuclei of SV40-transformed fibroblasts, while both leukemic cells andcolon cancer cells show higher enzyme activity than the equivalentnormal leukocytes and colon mucosa (Miwa, et al., Arch. Biochem.Biophys., 181, 313-321 (1977); Burzio, et al., Proc. Soc. Exp. Bioi.Med., 149, 933-938 (1975); and Hirai, et al., Cancer Res., 43, 3441-3446(1983)).

A number of low-molecular-weight inhibitors of PARP have been used toelucidate the functional role of poly (ADP-ribosyl)ation in DNA repair.In cells treated with alkylating agents, the inhibition of PARP leads toa marked increase in DNA-strand breakage and cell killing (Durkacz, etal., Nature, 283, 593-596 (1980); Berger, N. A., Radiation Research,101, 4-14 (1985)).

Subsequently, such inhibitors have been shown to enhance the effects ofradiation response by suppressing the repair of potentially lethaldamage (Ben-Hur, et al., British Journal of Cancer, 49 (Suppl. VI),34-42 (1984); Schlicker, et al., Int. J. Radiat. Bioi., 75, 91-100(1999)). PARP inhibitors have been reported to be effective in radiosensitising hypoxic tumour cells (U.S. Pat. No. 5,032,617; U.S. Pat. No.5,215,738 and U.S. Pat. No. 5,041,653).

Furthermore, PARP knockout (PARP −/−) animals exhibit genomicinstability in response to alkylating agents and γ-irradiation (Wang, etal., Genes Dev., 9, 509-520 (1995); Menissier de Murcia, et al., Proc.Natl. Acad. Sci. USA, 94, 7303-7307 (1997)).

A role for PARP has also been demonstrated in certain vascular diseases,septic shock, ischaemic injury and neurotoxicity (Cantoni, et al.,Biochim. Biophys. Acta, 1014, 1-7 (1989); Szabo, et al., J. Clin.Invest., 100, 723-735 (1997)). Oxygen radical DNA damage that leads tostrand breaks in DNA, which are subsequently recognised by PARP, is amajor contributing factor to such disease states as shown by PARPinhibitor studies (Cosi, et al., J. Neurosci. Res., 39, 38-46 (1994);Said, et al., Proc. Natl. Acad. Sci. USA., 93, 4688-4692 (1996)). Morerecently, PARP has been demonstrated to play a role in the pathogenesisof haemorrhagic shock (Liaudet, et al., Proc. Natl. Acad. Sci. USA.,97(3), 10203-10208 (2000)).

It has also been demonstrated that efficient retroviral infection ofmammalian cells is blocked by the inhibition of PARP activity. Suchinhibition of recombinant retroviral vector infections was shown tooccur in various different cell types (Gaken, et al., J. Virology,70(6), 3992-4000 (1996)). Inhibitors of PARP have thus been developedfor the use in anti-viral therapies and in cancer treatment (WO91/18591).

Moreover, PARP inhibition has been speculated to delay the onset ofaging characteristics in human fibroblasts (Rattan and Clark, Biochem.Biophys. Res. Comm., 201(2), 665-672 (1994)). This may be related to therole that PARP plays in controlling telomere function (d'Adda diFagagna, et al., Nature Gen., 23(1), 76-80 (1999)).

Some of the present inventors have previously described (WO 02/36576) aclass of 1(2H)-phthalazinone compounds which act as PARP inhibitors. Thecompounds have the general formula:

where A and B together represent an optionally substituted, fusedaromatic ring and where R_(C) is represented by -L-R_(L). A large numberof examples are of the formula:

where R represent one or more optional substituents.

The present inventors have now discovered that compounds where R is of acertain nature exhibit surprising levels of inhibition of the activityof PARP, and/or of potentiation of tumour cells to radiotherapy andvarious chemotherapies.

Accordingly, the first aspect of the present invention provides acompound of the formula (I):

and isomers, salts, solvates, chemically protected forms, and prodrugsthereof wherein:A and B together represent an optionally substituted, fused aromaticring;X can be NR^(X) or CR^(X)R^(Y);if X═NR^(X) then n is 1 or 2 and if X═CR^(X)R^(Y) then n is 1; R^(X) isselected from the group consisting of H, optionally substituted C₁₋₂₀alkyl, C₅₋₂₀ aryl, C₃₋₂₀ heterocyclyl, amido, thioamido, ester, acyl,and sulfonyl groups;R^(Y) is selected from H, hydroxy, amino;or R^(X) and R^(Y) may together form a spiro-C₃₋₇ cycloalkyl orheterocyclyl group;R^(C1) and R^(C2) are independently selected from the group consistingof hydrogen and C₁₋₄ alkyl, or when X is CR^(X)R^(Y), R^(C1), R^(C2),R^(X) and R^(Y), together with the carbon atoms to which they areattached, may form an optionally substituted fused aromatic ring; andR¹ is selected from H and halo.

Therefore, if X is CR^(X)R^(Y), then n is 1 and the compound is offormula (Ia):

If X is NR^(X), and n is 1, the compound is of formula (Ib):

If X is NR^(X), and n is 2, the compound is of formula (Ic):

A second aspect of the present invention provides a pharmaceuticalcomposition comprising a compound of the first aspect and apharmaceutically acceptable carrier or diluent.

A third aspect of the present invention provides the use of a compoundof the first aspect in a method of treatment of the human or animalbody.

A fourth aspect of the present invention provides the use of a compoundas defined in the first aspect of the invention in the preparation of amedicament for:

(a) inhibiting the activity of PARP (PARP-1 and/or PARP-2);(b) the treatment of: vascular disease; septic shock; ischaemic injury,both cerebral and cardiovascular; reperfusion injury, both cerebral andcardiovascular; neurotoxicity, including acute and chronic treatmentsfor stroke and Parkinsons disease; haemorraghic shock; inflammatorydiseases, such as arthritis; multiple sclerosis; secondary effects ofdiabetes; as well as the acute treatment of cytoxicity followingcardiovascular surgery or diseases ameliorated by the inhibition of theactivity of PARP;(c) use as an adjunct in cancer therapy or for potentiating tumour cellsfor treatment with ionizing radiation or chemotherapeutic agents.

In particular, compounds as defined in the first aspect of the inventioncan be used in anti-cancer combination therapies (or as adjuncts) alongwith alkylating agents, such as methyl methanesulfonate (MMS),temozolomide and dacarbazine (DTIC), also with topoisomerase-1inhibitors like Irinotecan, Rubitecan, Exatecan, Lurtotecan, Gimetecan,Diflomotecan (homocamptothecins); as well as 7-substitutednon-silatecans; the 7-silyl camptothecins, BNP 1350; andnon-camptothecin topoisomerase-I inhibitors such as indolocarbazolesalso dual topoisomerase-I and II inhibitors like the benzophenazines, XR11576/MLN 576 and benzopyridoindoles. Such combinations could be given,for example, as intravenous preparations or by oral administration asdependent on the preferred method of administration for the particularagent.

Other further aspects of the invention provide for the treatment ofdisease ameliorated by the inhibition of PARP, comprising administeringto a subject in need of treatment a therapeutically-effective amount ofa compound as defined in the first aspect, preferably in the form of apharmaceutical composition and the treatment of cancer, comprisingadministering to a subject in need of treatment atherapeutically-effective amount of a compound as defined in the firstaspect in combination, preferably in the form of a pharmaceuticalcomposition, simultaneously or sequentially with ionizing radiation orchemotherapeutic agents.

In further aspects of the present invention, the compounds may be usedin the preparation of a medicament for the treatment of cancer which isdeficient in Homologous Recombination (HR) dependent DNA double strandbreak (DSB) repair activity, or in the treatment of a patient with acancer which is deficient in HR dependent DNA DSB repair activity,comprising administering to said patient a therapeutically-effectiveamount of the compound.

The HR dependent DNA DSB repair pathway repairs double-strand breaks(DSBs) in DNA via homologous mechanisms to reform a continuous DNA helix(K. K. Khanna and S. P. Jackson, Nat. Genet. 27(3): 247-254 (2001)). Thecomponents of the HR dependent DNA DSB repair pathway include, but arenot limited to, ATM (NM_000051), RAD51 (NM_002875), RAD51L1 (NM_002877),RAD51C (NM_002876), RAD51L3 (NM_002878), DMC1 (NM_007068), XRCC2(NM_005431), XRCC3 (NM_005432), RAD52 (NM_002879), RAD54L (NM_003579),RAD54B (NM_012415), BRCA1 (NM_007295), BRCA2 (NM_000059), RAD50(NM_005732), MRE11A (NM_005590) and NBS1 (NM_002485). Other proteinsinvolved in the HR dependent DNA DSB repair pathway include regulatoryfactors such as EMSY (Hughes-Davies, et al., Cell, 115, pp 523-535). HRcomponents are also described in Wood, et al., Science, 291, 1284-1289(2001).

A cancer which is deficient in HR dependent DNA DSB repair may compriseor consist of one or more cancer cells which have a reduced or abrogatedability to repair DNA DSBs through that pathway, relative to normalcells i.e. the activity of the HR dependent DNA DSB repair pathway maybe reduced or abolished in the one or more cancer cells.

The activity of one or more components of the HR dependent DNA DSBrepair pathway may be abolished in the one or more cancer cells of anindividual having a cancer which is deficient in HR dependent DNA DSBrepair. Components of the HR dependent DNA DSB repair pathway are wellcharacterised in the art (see for example, Wood, et al., Science, 291,1284-1289 (2001)) and include the components listed above.

In some preferred embodiments, the cancer cells may have a BRCA1 and/ora BRCA2 deficient phenotype i.e. BRCA1 and/or BRCA2 activity is reducedor abolished in the cancer cells. Cancer cells with this phenotype maybe deficient in BRCA1 and/or BRCA2, i.e. expression and/or activity ofBRCA1 and/or BRCA2 may be reduced or abolished in the cancer cells, forexample by means of mutation or polymorphism in the encoding nucleicacid, or by means of amplification, mutation or polymorphism in a geneencoding a regulatory factor, for example the EMSY gene which encodes aBRCA2 regulatory factor (Hughes-Davies, et al., Cell, 115, 523-535).

BRCA1 and BRCA2 are known tumour suppressors whose wild-type alleles arefrequently lost in tumours of heterozygous carriers (Jasin M., Oncogene,21(58), 8981-93 (2002); Tutt, et al., Trends Mol Med., 8(12), 571-6,(2002)). The association of BRCA1 and/or BRCA2 mutations with breastcancer is well-characterised in the art (Radice, P. J., Exp Clin CancerRes., 21(3 Suppl), 9-12 (2002)). Amplification of the EMSY gene, whichencodes a BRCA2 binding factor, is also known to be associated withbreast and ovarian cancer.

Carriers of mutations in BRCA1 and/or BRCA2 are also at elevated risk ofcancer of the ovary, prostate and pancreas.

In some preferred embodiments, the individual is heterozygous for one ormore variations, such as mutations and polymorphisms, in BRCA1 and/orBRCA2 or a regulator thereof. The detection of variation in BRCA1 andBRCA2 is well-known in the art and is described, for example in EP 699754, EP 705 903, Neuhausen, S. L. and Ostrander, E. A., Genet. Test, 1,75-83 (1992); Chappnis, P. O. and Foulkes, W. D., Cancer Treat Res, 107,29-59 (2002); Janatova M., et al., Neoplasma, 50(4), 246-50 (2003);Jancarkova, N., Ceska Gynekol., 68(1), 11-6 (2003)). Determination ofamplification of the BRCA2 binding factor EMSY is described inHughes-Davies, et al., Cell, 115, 523-535).

Mutations and polymorphisms associated with cancer may be detected atthe nucleic acid level by detecting the presence of a variant nucleicacid sequence or at the protein level by detecting the presence of avariant (i.e. a mutant or allelic variant) polypeptide.

Definitions

The term “aromatic ring” is used herein in the conventional sense torefer to a cyclic aromatic structure, that is, a cyclic structure havingdelocalised π-electron orbitals.

The aromatic ring fused to the main core, i.e. that formed by -A-B-, maybear further fused aromatic rings (resulting in, e.g. naphthyl oranthracenyl groups). The aromatic ring(s) may comprise solely carbonatoms, or may comprise carbon atoms and one or more heteroatoms,including but not limited to, nitrogen, oxygen, and sulfur atoms. Thearomatic ring(s) preferably have five or six ring atoms.

The aromatic ring(s) may optionally be substituted. If a substituentitself comprises an aryl group, this aryl group is not considered to bea part of the aryl group to which it is attached. For example, the groupbiphenyl is considered herein to be a phenyl group (an aryl groupcomprising a single aromatic ring) substituted with a phenyl group.Similarly, the group benzylphenyl is considered to be a phenyl group (anaryl group comprising a single aromatic ring) substituted with a benzylgroup.

In one group of preferred embodiments, the aromatic group comprises asingle aromatic ring, which has five or six ring atoms, which ring atomsare selected from carbon, nitrogen, oxygen, and sulfur, and which ringis optionally substituted. Examples of these groups include, but are notlimited to, benzene, pyrazine, pyrrole, thiazole, isoxazole, andoxazole. 2-Pyrone can also be considered to be an aromatic ring, but isless preferred.

If the aromatic ring has six atoms, then preferably at least four, oreven five or all, of the ring atoms are carbon. The other ring atoms areselected from nitrogen, oxygen and sulphur, with nitrogen and oxygenbeing preferred. Suitable groups include a ring with: no hetero atoms(benzene); one nitrogen ring atom (pyridine); two nitrogen ring atoms(pyrazine, pyrimidine and pyridazine); one oxygen ring atom (pyrone);and one oxygen and one nitrogen ring atom (oxazine).

If the aromatic ring has five ring atoms, then preferably at least threeof the ring atoms are carbon. The remaining ring atoms are selected fromnitrogen, oxygen and sulphur. Suitable rings include a ring with: onenitrogen ring atom (pyrrole); two nitrogen ring atoms (imidazole,pyrazole); one oxygen ring atom (furan); one sulphur ring atom(thiophene); one nitrogen and one sulphur ring atom (isothiazole,thiazole); and one nitrogen and one oxygen ring atom (isoxazole oroxazole).

The aromatic ring may bear one or more substituent groups at anyavailable ring position. These substituents are selected from halo,nitro, hydroxy, ether, thiol, thioether, amino, C₁₋₇ alkyl, C₃₋₂₀heterocyclyl and C₅₋₂₀ aryl. The aromatic ring may also bear one or moresubstituent groups which together form a ring. In particular these maybe of formula —(CH₂)_(m)— or —O—(CH₂)_(p)—O—, where m is 2, 3, 4 or 5and p is 1, 2 or 3.

Alkyl: The term “alkyl” as used herein, pertains to a monovalent moietyobtained by removing a hydrogen atom from a carbon atom of a hydrocarboncompound having from 1 to 20 carbon atoms (unless otherwise specified),which may be aliphatic or alicyclic, and which may be saturated orunsaturated (e.g. partially unsaturated, fully unsaturated). Thus, theterm “alkyl” includes the sub-classes alkenyl, alkynyl, cycloalkyl,cycloalkyenyl, cylcoalkynyl, etc., discussed below.

In the context of alkyl groups, the prefixes (e.g. C₁₋₄, C₁₋₇, C₁₋₂₀,C₂₋₇, C₃₋₇, etc.) denote the number of carbon atoms, or range of numberof carbon atoms. For example, the term “C₁₋₄ alkyl”, as used herein,pertains to an alkyl group having from 1 to 4 carbon atoms. Examples ofgroups of alkyl groups include C₁₋₄ alkyl (“lower alkyl”), C₁₋₇ alkyl,and C₁₋₂₀ alkyl. Note that the first prefix may vary according to otherlimitations; for example, for unsaturated alkyl groups, the first prefixmust be at least 2; for cyclic alkyl groups, the first prefix must be atleast 3; etc.

Examples of (unsubstituted) saturated alkyl groups include, but are notlimited to, methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl(C₅), hexyl (C₆), heptyl (C₇), octyl (C₈), nonyl (C₉), decyl (C₁₀),undecyl (C₁₁), dodecyl (C₁₂), tridecyl (C₁₃), tetradecyl (C₁₄),pentadecyl (C₁₅), and eicodecyl (C₂₀).

Examples of (unsubstituted) saturated linear alkyl groups include, butare not limited to, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl(C₄), n-pentyl (amyl) (C₅), n-hexyl (C₆), and n-heptyl (C₇).

Examples of (unsubstituted) saturated branched alkyl groups include, butare not limited to, iso-propyl (C₃), iso-butyl (C₄), sec-butyl (C₄),tert-butyl (C₄), iso-pentyl (C₅), and neo-pentyl (C₅).

Alkenyl: The term “alkenyl”, as used herein, pertains to an alkyl grouphaving one or more carbon-carbon double bonds. Examples of alkenylgroups include C₂₋₄ alkenyl, C₂₋₇ alkenyl, C₂₋₂₀ alkenyl.

Examples of (unsubstituted) unsaturated alkenyl groups include, but arenot limited to, ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃),2-propenyl (allyl, —CH—CH═CH₂), isopropenyl (1-methylvinyl,—C(CH₃)═CH₂), butenyl (C₄), pentenyl (C₅), and hexenyl (C₆).

Alkynyl: The term “alkynyl”, as used herein, pertains to an alkyl grouphaving one or more carbon-carbon triple bonds. Examples of alkynylgroups include C₂₋₄ alkynyl, C₂₋₇ alkynyl, C₂₋₂₀ alkynyl.

Examples of (unsubstituted) unsaturated alkynyl groups include, but arenot limited to, ethynyl (ethinyl, —C≡CH) and 2-propynyl (propargyl,—CH₂—C≡CH).

Cycloalkyl: The term “cycloalkyl”, as used herein, pertains to an alkylgroup which is also a cyclyl group; that is, a monovalent moietyobtained by removing a hydrogen atom from an alicyclic ring atom of acarbocyclic ring of a carbocyclic compound, which carbocyclic ring maybe saturated or unsaturated (e.g. partially unsaturated, fullyunsaturated), which moiety has from 3 to 20 carbon atoms (unlessotherwise specified), including from 3 to 20 ring atoms. Thus, the term“cycloalkyl” includes the sub-classes cycloalkenyl and cycloalkynyl.Preferably, each ring has from 3 to 7 ring atoms. Examples of groups ofcycloalkyl groups include C₃₋₂₀ cycloalkyl, C₃₋₁₅ cycloalkyl, C₃₋₁₀cycloalkyl, C₃₋₇ cycloalkyl.

Examples of cycloalkyl groups include, but are not limited to, thosederived from:

-   -   saturated monocyclic hydrocarbon compounds:        cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅),        cyclohexane (C₆), cycloheptane (C₇), methylcyclopropane (C₄),        dimethylcyclopropane (C₅), methylcyclobutane (C₅),        dimethylcyclobutane (C₆), methylcyclopentane (C₆),        dimethylcyclopentane (C₇), methylcyclohexane (C₇),        dimethylcyclohexane (C₈), menthane (C₁₀);    -   unsaturated monocyclic hydrocarbon compounds:        cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅),        cyclohexene (C₆), methylcyclopropene (C₄), dimethylcyclopropene        (C₅), methylcyclobutene (C₅), dimethylcyclobutene (C₆),        methylcyclopentene (C₆), dimethylcyclopentene (C₇),        methylcyclohexene (C₇), dimethylcyclohexene (C₈);    -   saturated polycyclic hydrocarbon compounds:        thujane (C₁₀), carane (C₁₀), pinane (C₁₀), bornane (C₁₀),        norcarane (C₇), norpinane (C₇), norbornane (C₇), adamantane        (C₁₀), decalin (decahydronaphthalene) (C₁₀);    -   unsaturated polycyclic hydrocarbon compounds:        camphene (C₁₀), limonene (C₁₀), pinene (C₁₀);    -   polycyclic hydrocarbon compounds having an aromatic ring:        indene (C₉), indane (e.g., 2,3-dihydro-1H-indene) (C₉),        tetraline (1,2,3,4-tetrahydronaphthalene) (C₁₀), acenaphthene        (C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene (C₁₅),        aceanthrene (C₁₆), cholanthrene (C₂₀).

Heterocyclyl: The term “heterocyclyl”, as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a ring atomof a heterocyclic compound, which moiety has from 3 to 20 ring atoms(unless otherwise specified), of which from 1 to 10 are ringheteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of whichfrom 1 to 4 are ring heteroatoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₃₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆heterocyclyl”, as usedherein, pertains to a heterocyclyl group having 5 or 6 ring atoms.Examples of groups of heterocyclyl groups include C₃₋₂₀ heterocyclyl,C₅₋₂₀ heterocyclyl, C₃₋₁₅ heterocyclyl, C₅₋₁₅ heterocyclyl, C₃₋₁₂heterocyclyl, C₅₋₁₂ heterocyclyl, C₃₋₁₀ heterocyclyl, C₅₋₁₀heterocyclyl, C₃₋₇ heterocyclyl, C₅₋₇ heterocyclyl, and C₅₋₆heterocyclyl.

Examples of monocyclic heterocyclyl groups include, but are not limitedto, those derived from:

N₁: azindine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole) (C₅),pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrrole or3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆), dihydropyridine(C₆), tetrahydropyridine (C₆), azepine (C₇);O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole(dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆),pyran (C₆), oxepin (C₇);S₁: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene) (C₅),thiane (tetrahydrothiopyran) (C₆), thiepane (C₇);O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇);O₃: trioxane (C₆);N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline(C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆);N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole(C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆),dihydrooxazine (C₆), oxazine (C₆);N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆);N₂O₁: oxadiazine (C₆);O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and,N₁O₁S₁: oxathiazine (C₆).

Examples of substituted (non-aromatic) monocyclic heterocyclyl groupsinclude those derived from saccharides, in cyclic form, for example,furanoses (C₅), such as arabinofuranose, lyxofuranose, ribofuranose, andxylofuranse, and pyranoses (C₆), such as allopyranose, altropyranose,glucopyranose, mannopyranose, gulopyranose, idopyranose,galactopyranose, and talopyranose.

Spiro-C₃₋₇ cycloalkyl or heterocyclyl: The term “spiro C₃₋₇ cycloalkylor heterocyclyl” as used herein, refers to a C₃₋₇ cycloalkyl or C₃₋₇heterocyclyl ring joined to another ring by a single atom common to bothrings.

C₅₋₂₀ aryl: The term “C₅₋₂₀ aryl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from an aromaticring atom of a C₅₋₂₀ aromatic compound, said compound having one ring,or two or more rings (e.g., fused), and having from 5 to 20 ring atoms,and wherein at least one of said ring(s) is an aromatic ring.Preferably, each ring has from 5 to 7 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups” inwhich case the group may conveniently be referred to as a “C₅₋₂₀carboaryl” group.

Examples of C₅₋₂₀ aryl groups which do not have ring heteroatoms (i.e.C₅₋₂₀ carboaryl groups) include, but are not limited to, those derivedfrom benzene (i.e. phenyl) (C₆), naphthalene (C₁₀), anthracene (C₁₄),phenanthrene (C₁₄), and pyrene (C₁₆).

Alternatively, the ring atoms may include one or more heteroatoms,including but not limited to oxygen, nitrogen, and sulfur, as in“heteroaryl groups”. In this case, the group may conveniently bereferred to as a “C₅₋₂₀ heteroaryl” group, wherein “C₅₋₂₀” denotes ringatoms, whether carbon atoms or heteroatoms. Preferably, each ring hasfrom 5 to 7 ring atoms, of which from 0 to 4 are ring heteroatoms.

Examples of C₅₋₂₀ heteroaryl groups include, but are not limited to, C₅heteroaryl groups derived from furan (oxole), thiophene (thiole),pyrrole (azole), imidazole (1,3-diazole), pyrazole (1,2-diazole),triazole, oxazole, isoxazole, thiazole, isothiazole, oxadiazole,tetrazole and oxatriazole; and C₆ heteroaryl groups derived fromisoxazine, pyridine (azine), pyridazine (1,2-diazine), pyrimidine(1,3-diazine; e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine)and triazine.

The heteroaryl group may be bonded via a carbon or hetero ring atom.

Examples of C₅₋₂₀ heteroaryl groups which comprise fused rings, include,but are not limited to, C₉ heteroaryl groups derived from benzofuran,isobenzofuran, benzothiophene, indole, isoindole; C₁₀ heteroaryl groupsderived from quinoline, isoquinoline, benzodiazine, pyridopyridine; C₁₄heteroaryl groups derived from acridine and xanthene.

The above alkyl, heterocyclyl, and aryl groups, whether alone or part ofanother substituent, may themselves optionally be substituted with oneor more groups selected from themselves and the additional substituentslisted below.

Halo: —F, —Cl, —Br, and —I.

Hydroxy: —OH.

Ether: —OR, wherein R is an ether substituent, for example, a C₁₋₇ alkylgroup (also referred to as a C₁₋₇ alkoxy group), a C₃₋₂₀ heterocyclylgroup (also referred to as a C₃₋₂₀ heterocyclyloxy group), or a C₅₋₂₀aryl group (also referred to as a C₅₋₂₀ aryloxy group), preferably aC₁₋₇ alkyl group.

Nitro: —NO₂.

Cyano (nitrile, carbonitrile): —CN.

Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, H,a C₁₋₇ alkyl group (also referred to as C₁₋₇ alkylacyl or C₁₋₇alkanoyl), a C₃₋₂₀ heterocyclyl group (also referred to as C₃₋₂₀heterocyclylacyl), or a C₅₋₂₀ aryl group (also referred to as C₅₋₂₀arylacyl), preferably a C₁₋₇ alkyl group. Examples of acyl groupsinclude, but are not limited to, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃(propionyl), —C(═O)C(CH₃)₃ (butyryl), and —C(═O)Ph (benzoyl, phenone).

Carboxy (carboxylic acid): —COOH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR,wherein R is an ester substituent, for example, a C₁₋₇ alkyl group, aC₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ ar

yl group, preferably a C₁₋₇ alkyl group. Examples of ester groupsinclude, but are not limited to, —C(═O)OCH₃, —C(═O)OCH₂CH₃,—C(═O)OC(CH₃)₃, and —C(═O)OPh.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of amido groups include, but are not limited to,—C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and—C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², togetherwith the nitrogen atom to which they are attached, form a heterocyclicstructure as in, for example, piperidinocarbonyl, morpholinocarbonyl,thiomorpholinocarbonyl, and piperazinylcarbonyl.

Amino: —NR¹R², wherein R¹ and R² are independently amino substituents,for example, hydrogen, a C₁₋₇ alkyl group (also referred to as C₁₋₇alkylamino or di-C₁₋₇ alkylamino), a C₃₋₂₀ heterocyclyl group, or aC₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group, or, in the case ofa “cyclic” amino group, R¹ and R², taken together with the nitrogen atomto which they are attached, form a heterocyclic ring having from 4 to 8ring atoms. Examples of amino groups include, but are not limited to,—NH₂, —NHCH₃, —NHCH(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples ofcyclic amino groups include, but are not limited to, aziridinyl,azetidinyl, pyrrolidinyl, piperidino, piperazinyl, perhydrodiazepinyl,morpholino, and thiomorpholino. In particular, the cyclic amino groupsmay be substituted on their ring by any of the substituents definedhere, for example carboxy, carboxylate and amido.

Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amide substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group, mostpreferably H, and R² is an acyl substituent, for example, a C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably aC₁₋₇ alkyl group. Examples of acylamide groups include, but are notlimited to, —NHC(═O)CH₃, —NHC(═O)CH₂CH₃, and —NHC(═O)Ph. R¹ and R² maytogether form a cyclic structure, as in, for example, succinimidyl,maleimidyl, and phthalimidyl:

Ureido: —N(R¹)CONR²R³ wherein R² and R³ are independently aminosubstituents, as defined for amino groups, and R1 is a ureidosubstituent, for example, hydrogen, a C₁₋₇alkyl group, aC₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably hydrogen or aC₁₋₇alkyl group. Examples of ureido groups include, but are not limitedto, —NHCONH₂, —NHCONHMe, —NHCONHEt, —NHCONMe₂, —NHCONEt₂, —NMeCONH₂,—NMeCONHMe, —NMeCONHEt, —NMeCONMe₂, —NMeCONEt₂ and —NHCONHPh.

Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent,for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably a C₁₋₇ alkyl group. Examples of acyloxy groupsinclude, but are not limited to, —OC(═O)CH₃ (acetoxy), —OC(═O)CH₂CH₃,—OC(═O)C(CH₃)₃, —OC(═O)Ph, —OC(═O)C₆H₄F, and —OC(═O)CH₂Ph.

Thiol: —SH.

Thioether (sulfide): —SR, wherein R is a thioether substituent, forexample, a C₁₋₇ alkyl group (also referred to as a C₁₋₇ alkylthiogroup), a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably aC₁₋₇ alkyl group. Examples of C₁₋₇ alkylthio groups include, but are notlimited to, —SCH₃ and —SCH₂CH₃.

Sulfoxide (sulfinyl): —S(═O)R, wherein R is a sulfoxide substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfoxide groupsinclude, but are not limited to, —S(═O)CH₃ and —S(═O)CH₂CH₃.

Sulfonyl (sulfone): —S(═O)₂R, wherein R is a sulfone substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfone groupsinclude, but are not limited to, —S(═O)₂CH₃ (methanesulfonyl, mesyl),—S(═O)₂CF₃, —S(═O)₂CH₂CH₃, and 4-methylphenylsulfonyl (tosyl).

Thioamido (thiocarbamyl): —C(═S)NR¹R², wherein R¹ and R² areindependently amino substituents, as defined for amino groups. Examplesof amido groups include, but are not limited to, —C(═S)NH₂, —C(═S)NHCH₃,—C(═S)N(CH₃)₂, and —C(═S)NHCH₂CH₃.

Sulfonamino: —NR¹S(═O)₂R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfonamino substituent, for example, aC₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group,preferably a C₁₋₇alkyl group. Examples of sulfonamino groups include,but are not limited to, —NHS(═O)₂CH₃, —NHS(═O)₂Ph and —N(CH₃)S(═O)₂C₆H₅.

As mentioned above, the groups that form the above listed substituentgroups, e.g. C₁₋₇ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl, maythemselves be substituted. Thus, the above definitions cover substituentgroups which are substituted.

BRIEF DESCRIPTION OF FIGURES

FIGS. 1A-B show clonogenic survival curves of cells proficient anddeficient in Braca 1 exposed to compound (4) of the present invention.

FIG. 1A shows Brca1 wild type (11CO:▪), heterozygous (Cre6:▴) anddeficient (Cre10:●) embryonic fibroblastic stem (ES) cells undercontinuous exposure to compound 4. Error bars represent standard errorsof the mean.

FIG. 1B shows Brca2 wild type (D3:▪), heterozygous (Cre6:▴) anddeficient (Cre24: ●) ES cells under continuous exposure to compound 4.Error bars represent standard errors of the mean.

FIG. 2 shows an analysis of the effects of a compound of the invention(4) in another cell line lacking BRCA2 function in comparison to a BRCA2complemented line. Data shown is clonogenic survival of Brca2 deficient(V-C8:▪) and complemented (V-C8 BAC+: ▴) cells under continuous exposureto compound 4 at varying concentrations.

FURTHER PREFERENCES

The following preferences can apply to each aspect of the presentinvention, where applicable.

In the present invention, the fused aromatic ring(s) represented by-A-B- preferably consist of solely carbon ring atoms, and thus may bebenzene, naphthalene, and is more preferably benzene. As describedabove, these rings may be substituted, but in some embodiments arepreferably unsubstituted.

If the fused aromatic ring represented by -A-B- bears a substituentgroup, it is preferably attached to the atom which itself is attached tothe central ring meta- to the carbonyl group. Thus, if the fusedaromatic ring is a benzene ring, the preferred place of substitution isshown in the formula below by *:

which is usually termed the 5-position of the phthalazinone moiety.

R¹ is preferably selected from H, C1 and F, and is more preferably F.

It is preferred that R^(C1) and R^(C2) are independently selected fromhydrogen and C₁₋₄ alkyl, and more preferably H and methyl. It is morepreferred that at least one of R^(C1) and R^(C2) are hydrogen, with themost preferred option being that both are hydrogen.

When n is 2, X is NR^(X). In these embodiments, R^(X) is preferablyselected from the group consisting of: H; optionally substituted C₁₋₂₀alkyl (for example, optionally substituted C₅₋₂₀ arylmethyl); optionallysubstituted C₅₋₂₀ aryl; optionally substituted ester groups, wherein theester substituent is preferably C₁₋₂₀ alkyl; optionally substituted acylgroups; optionally substituted amido groups; optionally substitutedthioamido groups; and optionally substituted sulfonyl groups. R^(X) ismore preferably selected from the group consisting of: H; optionallysubstituted C₁₋₂₀ alkyl; optionally substituted C₅₋₂₀ aryl; andoptionally substituted ester groups, wherein the ester substituent ispreferably C₁₋₂₀ alkyl.

When n is I, X may be NR^(X) or CR^(X)CR^(Y).

In embodiments where X is NR^(X), R^(X) is preferably selected from thegroup consisting of: H; optionally substituted C₁₋₂₀ alkyl (for example,optionally substituted C₅₋₂₀ arylmethyl); optionally substituted C₅₋₂₀aryl; optionally substituted acyl; optionally substituted sulfonyl;optionally substituted amido; and optionally substituted thioamidogroups.

In embodiments where X is CR^(X)R^(Y), R^(Y) is preferably H. R^(X) ispreferably selected from the group consisting of: H; optionallysubstituted C₁₋₂₀ alkyl (for example, optionally substituted C₅₋₂₀arylmethyl); optionally substituted C₅₋₂₀ aryl; optionally substitutedC₃₋₂₀ heterocyclyl; optionally substituted acyl, wherein the acylsubstituent is preferably selected from C₅₋₂₀ aryl and C₃₋₂₀ heterocylyl(e.g. piperazinyl); optionally substituted amido, wherein the aminogroups are preferably selected from H and C₁₋₂₀ alkyl or together withthe nitrogen atom, form a C₅₋₂₀ heterocyclic group; and optionallysubstituted ester groups, wherein the ester substituent is preferablyselected from C₁₋₂₀ alkyl groups.

Particularly preferred compounds include: 1, 2, 3, 4, 10, 20, 59, 80,135, 146, 192, 194, 195, 211 and 212.

Where appropriate, the above preferences may be taken in combinationwith each other.

Includes Other Forms

Included in the above are the well known ionic, salt, solvate, andprotected forms of these substituents. For example, a reference tocarboxylic acid (—COOH) also includes the anionic (carboxylate) form(—COO⁻), a salt or solvate thereof, as well as conventional protectedforms. Similarly, a reference to an amino group includes the protonatedform (—N⁺HR¹R²), a salt or solvate of the amino group, for example, ahydrochloride salt, as well as conventional protected forms of an aminogroup. Similarly, a reference to a hydroxyl group also includes theanionic form (—O⁻), a salt or solvate thereof, as well as conventionalprotected forms of a hydroxyl group.

Isomers, Salts, Solvates, Protected Forms, and Prodrugs

Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diasterioisomeric, epimeric, stereoisomeric,tautomeric, conformational, or anomeric forms, including but not limitedto, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- andexo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+)and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms;synclinal- and anticlinal-forms; α- and β-forms; axial and equatorialforms; boat-, chair-, twist-, envelope-, and halfchair-forms; andcombinations thereof, hereinafter collectively referred to as “isomers”(or “isomeric forms”).

If the compound is in crystalline form, it may exist in a number ofdifferent polymorphic forms.

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers”, as used herein, are structural (orconstitutional) isomers (i.e. isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group,—CH₂O—CH₂OH. Similarly, a reference to ortho-chlorophenyl is not to beconstrued as a reference to its structural isomer, meta-chlorophenyl.However, a reference to a class of structures may well includestructurally isomeric forms falling within that class (e.g., C₁₋₇ alkylincludes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, andtert-butyl; methoxyphenyl includes ortho-, meta-, andpara-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol, imine/enamine, amide/imino alcohol,amidine/amidine, nitroso/oxime, thioketone/enethiol,N-nitroso/hyroxyazo, and nitro/aci-nitro.

Particularly relevant to the present invention is the tautomeric pairillustrated below:

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.asymmetric synthesis) and separation (e.g. fractional crystallisationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

Unless otherwise specified, a reference to a particular compound alsoincludes ionic, salt, solvate, and protected forms of thereof, forexample, as discussed below, as well as its different polymorphic forms.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge, et al., “PharmaceuticallyAcceptable Salts”, J. Pharm. Sci., 66, 1-19 (1977).

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g., —COOH may be —COO⁻), then a salt may be formedwith a suitable cation. Examples of suitable inorganic cations include,but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkalineearth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺.Examples of suitable organic cations include, but are not limited to,ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g., NH₃R⁺,NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammoniumions are those derived from: ethylamine, diethylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g., —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous. Examples of suitable organicanions include, but are not limited to, those derived from the followingorganic acids: acetic, propionic, succinic, gycolic, stearic, palmitic,lactic, malic, pamoic, tartaric, citric, gluconic, ascorbic, maleic,hydroxymaleic, phenylacetic, glutamic, aspartic, benzoic, cinnamic,pyruvic, salicyclic, sulfanilic, 2-acetyoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethanesulfonic, ethane disulfonic,oxalic, isethionic, valeric, and gluconic. Examples of suitablepolymeric anions include, but are not limited to, those derived from thefollowing polymeric acids: tannic acid, carboxymethyl cellulose.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

It may be convenient or desirable to prepare, purify, and/or handle theactive compound in a chemically protected form. The term “chemicallyprotected form,” as used herein, pertains to a compound in which one ormore reactive functional groups are protected from undesirable chemicalreactions, that is, are in the form of a protected or protecting group(also known as a masked or masking group or a blocked or blockinggroup). By protecting a reactive functional group, reactions involvingother unprotected reactive functional groups can be performed, withoutaffecting the protected group; the protecting group may be removed,usually in a subsequent step, without substantially affecting theremainder of the molecule. See, for example, “Protective Groups inOrganic Synthesis” (T. Green and P. Wuts; 3rd Edition; John Wiley andSons, 1999).

For example, a hydroxy group may be protected as an ether (—OR) or anester (—OC(═O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl(diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl ort-butyldimethylsilyl ether; or an acetyl ester (—OC(═O)CH₃, —OAc).

For example, an aldehyde or ketone group may be protected as an acetalor ketal, respectively, in which the carbonyl group (>C═O) is convertedto a diether (>C(OR)₂), by reaction with, for example, a primaryalcohol. The aldehyde or ketone group is readily regenerated byhydrolysis using a large excess of water in the presence of acid.

For example, an amine group may be protected, for example, as an amideor a urethane, for example, as: a methyl amide (—NHCO—CH₃); a benzyloxyamide (—NHCO—OCH₂C₆H₅, —NH-Cbz); as a t-butoxy amide (—NHCO—OC(CH₃)₃,—NH-Boc); a 2-biphenyl-2-propoxy amide (—NHCO—OC(CH₃)₂C₆H₄C₆H₅,—NH-Bpoc), as a 9-fluorenylmethoxy amide (—NH-Fmoc), as a6-nitroveratryloxy amide (—NH-Nvoc), as a 2-trimethylsilylethyloxy amide(—NH-Teoc), as a 2,2,2-trichloroethyloxy amide (—NH-Troc), as anallyloxy amide (—NH-Alloc), as a 2(-phenylsulphonyl)ethyloxy amide(—NH-Psec); or, in suitable cases, as an N-oxide (>NO.).

For example, a carboxylic acid group may be protected as an ester forexample, as: a C₁₋₇ alkyl ester (e.g. a methyl ester; a t-butyl ester);a C₁₋₇ haloalkyl ester (e.g. a C₁₋₇ trihaloalkyl ester); a triC₁₋₇alkylsilyl-C₁₋₇ alkyl ester; or a C₅₋₂₀ aryl-C₁₋₇ alkyl ester (e.g. abenzyl ester; a nitrobenzyl ester); or as an amide, for example, as amethyl amide.

For example, a thiol group may be protected as a thioether (—SR), forexample, as: a benzyl thioether; an acetamidomethyl ether(—S—CH₂NHC(═O)CH₃).

It may be convenient or desirable to prepare, purify, and/or handle theactive compound in the form of a prodrug. The term “prodrug”, as usedherein, pertains to a compound which, when metabolised (e.g. in vivo),yields the desired active compound. Typically, the prodrug is inactive,or less active than the active compound, but may provide advantageoushandling, administration, or metabolic properties.

For example, some prodrugs are esters of the active compound (e.g. aphysiologically acceptable metabolically labile ester). Duringmetabolism, the ester group (—C(═O)OR) is cleaved to yield the activedrug. Such esters may be formed by esterification, for example, of anyof the carboxylic acid groups (—C(═O)OH) in the parent compound, with,where appropriate, prior protection of any other reactive groups presentin the parent compound, followed by deprotection if required. Examplesof such metabolically labile esters include, but are not limited to,those wherein R is C₁₋₂₀ alkyl (e.g. -Me, -Et); C₁₋₇ aminoalkyl (e.g.aminoethyl; 2-(N,N-diethylamino)ethyl; 2-(4-morpholino)ethyl); andacyloxy-C₁₋₇ alkyl (e.g. acyloxymethyl; acyloxyethyl; e.g.pivaloyloxymethyl; acetoxymethyl; 1-acetoxyethyl;1-(1-methoxy-1-methyl)ethyl-carbonxyloxyethyl; 1-(benzoyloxy)ethyl;isopropoxy-carbonyloxymethyl; 1-isopropoxy-carbonyloxyethyl;cyclohexyl-carbonyloxymethyl; 1-cyclohexyl-carbonyloxyethyl;cyclohexyloxy-carbonyloxymethyl; 1-cyclohexyloxy-carbonyloxyethyl;(4-tetrahydropyranyloxy) carbonyloxymethyl;1-(4-tetrahydropyranyloxy)carbonyloxyethyl;(4-tetrahydropyranyl)carbonyloxymethyl; and1-(4-tetrahydropyranyl)carbonyloxyethyl).

Further suitable prodrug forms include phosphonate and glycolate salts.In particular, hydroxy groups (—OH), can be made into phosphonateprodrugs by reaction with chlorodibenzylphosphite, followed byhydrogenation, to form a phosphonate group —O—P(═O)(OH)₂. Such a groupcan be cleaved by phosphatase enzymes during metabolism to yield theactive drug with the hydroxy group.

Also, some prodrugs are activated enzymatically to yield the activecompound, or a compound which, upon further chemical reaction, yieldsthe active compound. For example, the prodrug may be a sugar derivativeor other glycoside conjugate, or may be an amino acid ester derivative.

Acronyms

For convenience, many chemical moieties are represented using well knownabbreviations, including but not limited to, methyl (Me), ethyl (Et),n-propyl (nPr), iso-propyl (iPr), n-butyl (nBu), tert-butyl (tBu),n-hexyl (nHex), cyclohexyl (cHex), phenyl (Ph), biphenyl (biPh), benzyl(Bn), naphthyl (naph), methoxy (MeO), ethoxy (EtO), benzoyl (Bz), andacetyl (Ac).

For convenience, many chemical compounds are represented using wellknown abbreviations, including but not limited to, methanol (MeOH),ethanol (EtOH), iso-propanol (i-PrOH), methyl ethyl ketone (MEK), etheror diethyl ether (Et₂O), acetic acid (AcOH), dichloromethane (methylenechloride, DCM), trifluoroacetic acid (TFA), dimethylformamide (DMF),tetrahydrofuran (THF), and dimethylsulfoxide (DMSO).

Synthesis

In the synthesis routes given below, the A-B fused ring is shown as afused benzene ring for convenience. Compounds in which the A-B ring isother than benzene may be synthesised using methodologies analogous tothose described below by the use of appropriate alternative startingmaterials.

Compounds of the present invention may be synthesised by reaction of acompound of Formula 1:

in which R¹ is as previously defined, with a compound of Formula 2:

in which n, R^(C1), R^(C2) and X are as previously defined, in thepresence of a coupling reagent system, for example2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate,2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphateor (dimethylaminopropyl)ethylcarbodiimidehydrochloride/hydroxybenzotriazole, in the presence of a base, forexample diisopropylethylamine, in a solvent, for exampledimethylacetamide or dichloromethane, at a temperature in the range of0° C. to the boiling point of the solvent used.

Alternatively, compounds of the present invention may be synthesised byconversion of a compound of Formula 1 into an activated species, forexample an acid chloride or an activated ester such as anN-hydroxysuccinimide ester, using well-known methodologies, and reactionof the activated species with a compound of Formula 2.

Compounds of Formula 1 may be synthesised by reaction of a compound ofFormula 3:

in which R¹ is as previously defined, or a compound of Formula 4:

in which R¹ is as previously defined, or a mixture of a compound ofFormula 3 and a compound of Formula 4, with a source of hydrazine, forexample hydrazine hydrate, optionally in the presence of a base, forexample triethylamine, optionally in the presence of a solvent, forexample industrial methylated spirit, at a temperature in the range of0° C. to the boiling point of the solvent used.

Compounds of Formula 3 or Formula 4, or mixtures thereof, may besynthesised by reaction of a compound of Formula 5:

in which R¹ is as previously defined, with a reagent capable ofhydrolysing a nitrile moiety, for example sodium hydroxide, in thepresence of a solvent, for example water, at a temperature in the rangeof 0° C. to the boiling point of the solvent used.

Compounds of Formula 5 may be synthesised by reaction of a compound ofFormula 6:

in which R¹ is as previously defined, with a compound of Formula 7:

in the presence of a base, for example sodium methoxide, in a solvent,for example methanol, optionally in the presence of a water scavenger,for example ethyl propionate, at a temperature in the range of 0° C. tothe boiling point of the solvent used.

Compounds of Formula 1 may also be synthesised by reaction of a compoundof Formula 8:

in which R¹ is as previously defined, with a reagent capable ofhydrolysing a nitrile moiety, for example sodium hydroxide, in thepresence of a solvent, for example water, at a temperature in the rangeof 0° C. to the boiling point of the solvent used, followed by reactionof the resulting intermediate with a source of hydrazine, for examplehydrazine hydrate, at a temperature in the range of 0° C. to the boilingpoint of the solvent used.

Compounds of Formula 8 may be synthesised by reaction of a compound ofFormula 9:

in which R_(a) is a C₁₋₄ alkyl group, with a compound of Formula 6, inthe presence of a base, for example triethylamine or lithiumhexamethyldisilazide, in the presence of a solvent, for exampletetrahydrofuran, at a temperature in the range of −80° C. to the boilingpoint of the solvent used.

Compounds of Formula 9 may be synthesised by methods analogous to thosedescribed in WO 02/26576.

Compounds of Formula 1 may also be synthesised by methods analogous tothose described above in which the nitrile moiety in all Formulae isreplaced by other moieties capable of generating a carboxylic acid, forexample ester or carboxamide moieties.

Compounds of Formula 2 are commercially available or may be synthesisedby methods reported in the chemical literature.

Compounds of the present invention in which X is CR^(X)R^(Y), in whichone of R^(X) or R^(Y) is an amido moiety, and which may therefore berepresented by Formula 10:

in which n, R^(C1), R^(C2), R¹ and R^(X) are as previously defined andR^(N1) and R^(N2) are each individually selected from the groupconsisting of H, optionally substituted C₁₋₂₀ alkyl, C₅₋₂₀ aryl, C₃₋₂₀heterocyclyl, or may together form an optionally substituted C₃₋₇cycloalkyl or heterocyclyl group, may be synthesised by reaction of acompound of Formula 11:

in which n, R^(C1), R^(C2), R¹ and R^(X) are as previously defined, witha compound of Formula HNR^(N1)R^(N2), in which R^(N1) and R^(N2) are aspreviously defined, in the presence of a coupling reagent system, forexample 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate or (dimethylaminopropyl)ethylcarbodiimidehydrochloride/hydroxybenzotriazole, in the presence of a base, forexample diisopropylethylamine, in a solvent, for exampledimethylacetamide or dichloromethane, at a temperature in the range of0° C. to the boiling point of the solvent used.

Alternatively, compounds of Formula 10 may be synthesised by conversionof a compound of Formula 11 into an activated species, for example anacid chloride or an activated ester such as an N-hydroxysuccinimideester, using well-known methodologies, and reaction of the activatedspecies with a compound of Formula HNR^(N1)R^(N2).

Compounds of Formula 11 may be synthesised by deprotection of aprotected form of a compound of Formula 11, for example a compound ofFormula 12:

in which n, R^(C1), R^(C2), R¹ and R^(X) are as previously defined andR⁰¹ is a C₁₋₄ alkyl group, using well known methodologies, for examplebase-catalysed hydrolysis in the presence of a source of hydroxide, forexample sodium or lithium hydroxide, in the presence of a solvent, forexample water and/or tetrahydrofuran, at a temperature in the range of0° C. to the boiling point of the solvent used.

Compounds of Formula 12 may be synthesised from compounds of Formula 1by the previously described methods.

Compounds of Formula HNR^(N1)R^(N2) are commercially available or may besynthesised by methods reported in the chemical literature.

Compounds of the present invention in which X is NH and which maytherefore be represented by Formula 13:

in which n, R^(C1), R^(C2) and R¹ are as previously defined, may besynthesised by deprotection of a protected form of a compound of Formula13, for example a compound of Formula 14:

in which n, R^(C1), R^(C2) and R¹ are as previously defined, using wellknown methodologies, for example acid-catalysed cleavage, in thepresence of an acid, for example trifluoroacetic acid or hydrochloricacid, in the presence of a solvent, for example dichloromethane orethanol and/or water, at a temperature in the range of 0° C. to theboiling point of the solvent used.

Compounds of Formula 14 may be synthesised from compounds of Formula 1by the previously described methods.

Compounds of the present invention in which X is NR^(X), in which R^(X)is an acyl moiety, and which may therefore be represented by Formula 15:

in which n, R^(C1), R^(C2) and R¹ are as previously defined and R^(C3)is selected from the group consisting of optionally substituted C₁₋₂₀alkyl, C₅₋₂₀ aryl and C₃₋₂₀ heterocyclyl, may be synthesised by reactionof a compound of Formula 13 with a compound of Formula R^(C3)COX, inwhich R^(C3) is as previously defined and X is a suitable leaving group,for example a halogen such as chloro, optionally in the presence of abase, for example pyridine, triethylamine or diisopropylethylamine,optionally in the presence of a solvent, for example dichloromethane, ata temperature in the range of 0° C. to the boiling point of the solventused.

Compounds of Formula R^(C3)COX are commercially available or may besynthesised by methods reported in the chemical literature.

Compounds of Formula 15 may also be synthesised by reaction of acompound of Formula 13 with a compound of Formula R^(C3)CO₂H, in whichR^(C3) is as previously defined, in the presence of a coupling reagentsystem, for example 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate or (dimethylaminopropyl)ethylcarbodiimidehydrochloride/hydroxybenzotriazole, in the presence of a base, forexample diisopropylethylamine, in a solvent, for exampledimethylacetamide or dichloromethane, at a temperature in the range of0° C. to the boiling point of the solvent used.

Compounds of Formula R^(C3)CO₂H are commercially available or may besynthesised by methods reported in the chemical literature.

Compounds of the present invention in which X is NR^(X), in which R^(X)is an amido or thioamido moiety, and which may therefore be representedby Formula 16:

in which n, R^(C1), R^(C2) and R¹ are as previously defined, Y is O or Sand R^(N3) is selected from the group consisting of optionallysubstituted C₁₋₂₀ alkyl, C₅₋₂₀ aryl and C₃₋₂₀ heterocyclyl, may besynthesised by reaction of a compound of Formula 13 with a compound ofFormula R^(N3)NCY, in which Y and R^(N3) are as previously defined, inthe presence of a solvent, for example dichloromethane, at a temperaturein the range of 0° C. to the boiling point of the solvent used.

Compounds of Formula R^(N3)NCY are commercially available or may besynthesised by methods reported in the chemical literature.

Compounds of the present invention in which X is NR^(X), in which R^(X)is a sulfonyl moiety, and which may therefore be represented by Formula17:

in which n, R^(C1), R^(C2) and R¹ are as previously defined and R^(S1)is selected from the group consisting of optionally substituted C₁₋₂₀alkyl, C₅₋₂₀ aryl and C₃₋₂₀ heterocyclyl, may be synthesised by reactionof a compound of Formula 13 with a compound of Formula R^(S1)SO₂Cl, inwhich R^(S1) is as previously defined, optionally in the presence of abase, for example pyridine, triethylamine or diisopropylethylamine, inthe presence of a solvent, for example dichloromethane, at a temperaturein the range of 0° C. to the boiling point of the solvent used.

Compounds of Formula R^(S1)SO₂Cl are commercially available or may besynthesised by methods reported in the chemical literature.

Compounds of the present invention in which X is NR^(X), in which R^(X)is selected from the group consisting of optionally substituted C₁₋₂₀alkyl or C₃₋₂₀ heterocyclyl, and which may therefore be represented byFormula 18:

in which n, R^(C1), R^(C2) and R¹ are as previously defined and R^(C4)and R^(C5) are each individually selected from the group consisting ofH, optionally substituted C₁₋₂₀ alkyl, C₅₋₂₀ aryl, C₃₋₂₀ heterocyclyl,or may together form an optionally substituted C₃₋₇ cycloalkyl orheterocyclyl group, may be synthesised by reaction of a compound ofFormula 13 with a compound of Formula R^(C4)COR^(C5), in which R^(C4)and R^(C5) are as previously defined, in the presence of a reducingagent, for example sodium cyanoborohydride or sodiumtriacetoxyborohydride, in the presence of a solvent, for examplemethanol, optionally in the presence of an acid catalyst, for exampleacetic acid, at a temperature in the range of 0° C. to the boiling pointof the solvent used.

Compounds of Formula R^(C4)COR^(C5) are commercially available or may besynthesised by methods reported in the chemical literature.

Use

The present invention provides active compounds, specifically, active ininhibiting the activity of PARP.

The term “active” as used herein, pertains to compounds which arecapable of inhibiting PARP activity, and specifically includes bothcompounds with intrinsic activity (drugs) as well as prodrugs of suchcompounds, which prodrugs may themselves exhibit little or no intrinsicactivity.

One assay which may conveniently be used in order to assess the PARPinhibition offered by a particular compound is described in the examplesbelow.

The present invention further provides a method of inhibiting theactivity of PARP in a cell, comprising contacting said cell with aneffective amount of an active compound, preferably in the form of apharmaceutically acceptable composition. Such a method may be practisedin vitro or in vivo.

For example, a sample of cells may be grown in vitro and an activecompound brought into contact with said cells, and the effect of thecompound on those cells observed. As examples of “effect”, the amount ofDNA repair effected in a certain time may be determined. Where theactive compound is found to exert an influence on the cells, this may beused as a prognostic or diagnostic marker of the efficacy of thecompound in methods of treating a patient carrying cells of the samecellular type.

The term “treatment”, as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g. in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, amelioration of the condition,and cure of the condition. Treatment as a prophylactic measure (i.e.prophylaxis) is also included.

The term “adjunct” as used herein relates to the use of active compoundsin conjunction with known therapeutic means. Such means includecytotoxic regimens of drugs and/or ionising radiation as used in thetreatment of different cancer types. In particular, the active compoundsare known to potentiate the actions of a number of cancer chemotherapytreatments, which include the topoisomerase class of poisons and most ofthe known alkylating agents used in treating cancer.

Active compounds may also be used as cell culture additives to inhibitPARP, for example, in order to sensitize cells to known chemotherapeuticagents or ionising radiation treatments in vitro.

Active compounds may also be used as part of an in vitro assay, forexample, in order to determine whether a candidate host is likely tobenefit from treatment with the compound in question.

Administration

The active compound or pharmaceutical composition comprising the activecompound may be administered to a subject by any convenient route ofadministration, whether systemically/peripherally or at the site ofdesired action, including but not limited to, oral (e.g. by ingestion);topical (including e.g. transdermal, intranasal, ocular, buccal, andsublingual); pulmonary (e.g. by inhalation or insufflation therapyusing, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal;parenteral, for example, by injection, including subcutaneous,intradermal, intramuscular, intravenous, intraarterial, intracardiac,intrathecal, intraspinal, intracapsular, subcapsular, intraorbital,intraperitoneal, intratracheal, subcuticular, intraarticular,subarachnoid, and intrasternal; by implant of a depot, for example,subcutaneously or intramuscularly.

The subject may be a eukaryote, an animal, a vertebrate animal, amammal, a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine(e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. ahorse), a primate, simian (e.g. a monkey or ape), a monkey (e.g.marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orangutang,gibbon), or a human.

Formulations

While it is possible for the active compound to be administered alone,it is preferable to present it as a pharmaceutical composition (e.g.,formulation) comprising at least one active compound, as defined above,together with one or more pharmaceutically acceptable carriers,adjuvants, excipients, diluents, fillers, buffers, stabilisers,preservatives, lubricants, or other materials well known to thoseskilled in the art and optionally other therapeutic or prophylacticagents.

Thus, the present invention further provides pharmaceuticalcompositions, as defined above, and methods of making a pharmaceuticalcomposition comprising admixing at least one active compound, as definedabove, together with one or more pharmaceutically acceptable carriers,excipients, buffers, adjuvants, stabilisers, or other materials, asdescribed herein.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgement, suitable for use in contactwith the tissues of a subject (e.g. human) without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Each carrier,excipient, etc. must also be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts. See, for example, “Handbook of PharmaceuticalAdditives”, 2nd Edition (eds. M. Ash and I. Ash), 2001 (SynapseInformation Resources, Inc., Endicott, N.Y., USA), “Remington'sPharmaceutical Sciences”, 20th edition, pub. Lippincott, Williams &Wilkins, 2000; and “Handbook of Pharmaceutical Excipients”, 2nd edition,1994.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Suchmethods include the step of bringing into association the activecompound with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active compound with liquidcarriers or finely divided solid carriers or both, and then if necessaryshaping the product.

Formulations may be in the form of liquids, solutions, suspensions,emulsions, elixirs, syrups, tablets, losenges, granules, powders,capsules, cachets, pills, ampoules, suppositories, pessaries, ointments,gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses,electuaries, or aerosols.

Formulations suitable for oral administration (e.g., by ingestion) maybe presented as discrete units such as capsules, cachets or tablets,each containing a predetermined amount of the active compound; as apowder or granules; as a solution or suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion; as a bolus; as an electuary; or as apaste.

A tablet may be made by conventional means, e.g. compression or molding,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing in a suitable machine the active compoundin a free-flowing form such as a powder or granules, optionally mixedwith one or more binders (e.g. povidone, gelatin, acacia, sorbitol,tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g.lactose, microcrystalline cellulose, calcium hydrogen phosphate);lubricants (e.g. magnesium stearate, talc, silica); disintegrants (e.g.sodium starch glycolate, cross-linked povidone, cross-linked sodiumcarboxymethyl cellulose); surface-active or dispersing or wetting agents(e.g., sodium lauryl sulfate); and preservatives (e.g., methylp-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid). Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active compound therein using,for example, hydroxypropylmethyl cellulose in varying proportions toprovide the desired release profile. Tablets may optionally be providedwith an enteric coating, to provide release in parts of the gut otherthan the stomach.

Formulations suitable for topical administration (e.g. transdermal,intranasal, ocular, buccal, and sublingual) may be formulated as anointment, cream, suspension, lotion, powder, solution, past, gel, spray,aerosol, or oil. Alternatively, a formulation may comprise a patch or adressing such as a bandage or adhesive plaster impregnated with activecompounds and optionally one or more excipients or diluents.

Formulations suitable for topical administration in the mouth includelosenges comprising the active compound in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activecompound in an inert basis such as gelatin and glycerin, or sucrose andacacia; and mouthwashes comprising the active compound in a suitableliquid carrier.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active compound is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the active compound.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of about 20 to about 500 microns which is administered in themanner in which snuff is taken, i.e. by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable formulations wherein the carrier is a liquid for administrationas, for example, nasal spray, nasal drops, or by aerosol administrationby nebuliser, include aqueous or oily solutions of the active compound.

Formulations suitable for administration by inhalation include thosepresented as an aerosol spray from a pressurised pack, with the use of asuitable propellant, such as dichlorodifluoromethane,trichlorofluoromethane, dichoro-tetrafluoroethane, carbon dioxide, orother suitable gases.

Formulations suitable for topical administration via the skin includeointments, creams, and emulsions. When formulated in an ointment, theactive compound may optionally be employed with either a paraffinic or awater-miscible ointment base. Alternatively, the active compounds may beformulated in a cream with an oil-in-water cream base. If desired, theaqueous phase of the cream base may include, for example, at least about30% w/w of a polyhydric alcohol, i.e., an alcohol having two or morehydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol,sorbitol, glycerol and polyethylene glycol and mixtures thereof. Thetopical formulations may desirably include a compound which enhancesabsorption or penetration of the active compound through the skin orother affected areas. Examples of such dermal penetration enhancersinclude dimethylsulfoxide and related analogues.

When formulated as a topical emulsion, the oily phase may optionallycomprise merely an emulsifier (otherwise known as an emulgent), or itmay comprises a mixture of at least one emulsifier with a fat or an oilor with both a fat and an oil. Preferably, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabiliser. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabiliser(s) make up theso-called emulsifying wax, and the wax together with the oil and/or fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

Suitable emulgents and emulsion stabilisers include Tween 60, Span 80,cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodiumlauryl sulphate. The choice of suitable oils or fats for the formulationis based on achieving the desired cosmetic properties, since thesolubility of the active compound in most oils likely to be used inpharmaceutical emulsion formulations may be very low. Thus the creamshould preferably be a non-greasy, non-staining and washable productwith suitable consistency to avoid leakage from tubes or othercontainers. Straight or branched chain, mono- or dibasic alkyl esterssuch as di-isoadipate, isocetyl stearate, propylene glycol diester ofcoconut fatty acids, isopropyl myristate, decyl oleate, isopropylpalmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branchedchain esters known as Crodamol CAP may be used, the last three beingpreferred esters. These may be used alone or in combination depending onthe properties required. Alternatively, high melting point lipids suchas white soft paraffin and/or liquid paraffin or other mineral oils canbe used.

Formulations suitable for rectal administration may be presented as asuppository with a suitable base comprising, for example, cocoa butteror a salicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active compound, such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration (e.g., by injection,including cutaneous, subcutaneous, intramuscular, intravenous andintradermal), include aqueous and non-aqueous isotonic, pyrogen-free,sterile injection solutions which may contain anti-oxidants, buffers,preservatives, stabilisers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. Examples of suitable isotonic vehicles for use insuch formulations include Sodium Chloride Injection, Ringer's Solution,or Lactated Ringer's Injection. Typically, the concentration of theactive compound in the solution is from about 1 ng/ml to about 10 μg/ml,for example from about 10 ng/ml to about 1 μg/ml. The formulations maybe presented in unit-dose or multi-dose sealed containers, for example,ampoules and vials, and may be stored in a freeze-dried (lyophilised)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets. Formulations may be in the form ofliposomes or other microparticulate systems which are designed to targetthe active compound to blood components or one or more organs.

Dosage

It will be appreciated that appropriate dosages of the active compounds,and compositions comprising the active compounds, can vary from patientto patient. Determining the optimal dosage will generally involve thebalancing of the level of therapeutic benefit against any risk ordeleterious side effects of the treatments of the present invention. Theselected dosage level will depend on a variety of factors including, butnot limited to, the activity of the particular compound, the route ofadministration, the time of administration, the rate of excretion of thecompound, the duration of the treatment, other drugs, compounds, and/ormaterials used in combination, and the age, sex, weight, condition,general health, and prior medical history of the patient. The amount ofcompound and route of administration will ultimately be at thediscretion of the physician, although generally the dosage will be toachieve local concentrations at the site of action which achieve thedesired effect without causing substantial harmful or deleteriousside-effects.

Administration in vivo can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician.

In general, a suitable dose of the active compound is in the range ofabout 100 μg to about 250 mg per kilogram body weight of the subject perday. Where the active compound is a salt, an ester, prodrug, or thelike, the amount administered is calculated on the basis of the parentcompound and so the actual weight to be used is increasedproportionately.

Synthesis Data General Experimental Methods Preparative HPLC

Samples were purified with a Waters mass-directed purification systemutilising a Waters 600 LC pump, Waters Xterra C18 column (5 μm 19 mm×50mm) and Micromass ZQ mass spectrometer, operating in positive ionelectrospray ionisation mode. Mobile phases A (0.1% formic acid inwater) and B (0.1% formic acid in acetonitrile) were used in a gradient;5% B to 100% over 7 min, held for 3 min, at a flow rate of 20 ml/min.

Analytical HPLC-MS

Analytical HPLC was typically carried out with a Spectra System P4000pump and Jones Genesis C18 column (4 μm, 50 mm×4.6 mm). Mobile phases A(0.1% formic acid in water) and B (acetonitrile) were used in a gradientof 5% B for 1 min rising to 98% B after 5 min, held for 3 min at a flowrate of 2 ml/min. Detection was by a TSP UV 6000LP detector at 254 nm UVand range 210-600 nm PDA. The Mass spectrometer was a Finnigan LCQoperating in positive ion electrospray mode.

NMR

¹H NMR and ¹³C NMR were typically recorded using Bruker DPX 300spectrometer at 300 MHz and 75 MHz respectively. Chemical shifts werereported in parts per million (ppm) on the δ scale relative totetramethylsilane internal standard. Unless stated otherwise all sampleswere dissolved in DMSO-d₆.

Synthesis of Key Intermediates a.3-(4-Oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (A)

A mixture of 27% sodium methoxide solution in methanol (400 g, 2 mol)and methanol (150 ml) was added dropwise between ambient temperature and30° C. over 15 minutes to a stirred mixture of phthalide (67 g, 0.5mol), 3-formylbenzonitrile (65.5 g, 0.5 mol) and ethyl propionate (250ml), the mixture was stirred at ambient temperature for 40 minutes andat reflux temperature for 1 hour, then it was allowed to cool to ambienttemperature. The resulting red solid was collected by filtration, washedwith ethyl acetate (2×50 ml) and dissolved in water (1800 ml). Thesolution was acidified by the addition of acetic acid (60 ml) and theresulting red solid was collected by filtration, washed with water(2×200 ml) and dried in vacuo to give3-(1,3-dioxoindan-2-yl)benzonitrile (83.2 g) as a dark red solid, m.pt.179-182° C., m/z (M+H)^(+.) 248, which was used without furtherpurification.

3-(1,3-Dioxoindan-2-yl)benzonitrile (74.18 g, 0.3 mol) was added inportions to a solution of sodium hydroxide (36 g, 0.9 mol) in water (580ml), the resulting dark red suspension was stirred at reflux temperaturefor 5 hours, then it was cooled to ambient temperature and washed withethyl acetate (3×300 ml). The aqueous solution was acidified by thedropwise addition of concentrated hydrochloric acid (110 ml), themixture was stirred at ambient temperature for 1 hour, then theresulting solid was collected by filtration, washed with water (2×200ml) and dried in vacuo to give a 1:1 mixture of3-(1,3-dioxoindan-2-yl)benzoic acid, (M+H)^(+.) 267, and2-[2-(3-carboxyphenyl)acetyl]benzoic acid, (M+H)^(+.) 285, (69.32 g),which was used without further purification.

The mixture obtained in the previous step (52.8 g) was added to asolution of triethylamine (37.55 g, 0.372 mol) in industrial methylatedspirit (500 ml) and the resulting cloudy solution was filtered through apad of filter-aid to give a clear solution. Hydrazine monohydrate (9.3g, 0.186 mol) was added in one portion at ambient temperature, thestirred mixture was heated under reflux for 1 hour, then it wasconcentrated in vacuo to approximately 250 ml and added to a solution ofsodium acetate (41 g, 0.5 mol) in water (500 ml). The mixture wasbrought to pH 7 by the dropwise addition of concentrated hydrochloricacid, then it was stirred at ambient temperature for 3 hours. Theresulting solid was collected by filtration, washed with water (50 ml)and dried in vacuo to give a white solid (15.62 g). The combinedfiltrate and washings were acidified to pH 6 by the addition ofhydrochloric acid, then the mixture was stirred at ambient temperaturefor 3 hours. The resulting solid was collected by filtration, washedwith water (50 ml) and dried in vacuo to give a second crop of off-whitesolid (17.57 g). The combined filtrate and washings from the second cropwere readjusted to pH 6 and treated as before to give a third crop ofpale orange solid (6.66 g). The three crops were combined to giveessentially pure 3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid(A), (M+H)^(+.) 281, δ_(H) 4.4 (2H, s), 7.2-7.4 (1H, m), 7.5-7.6 (1H,m), 7.7-8.0 (5H, m), 8.1-8.2 (1H, m), 12.6 (1H, s)

b. 2-Fluoro-5-(4-oxo-3,4-dihydro-phthalazin-1-ylmethyl)benzoic acid (B)

Dimethyl phosphite (22.0 g, 0.2 mol) was added drop-wise to a solutionof sodium methoxide (43.0 g) in methanol (100 ml) at 0° C.2-Carboxybenzaldehyde (21.0 g, 0.1 mol) was then added portion-wise tothe reaction mixture as a slurry in methanol (40 ml), with thetemperature kept below 5° C. The resulting pale yellow solution waswarmed to 20° C. over 1 hour. Methanesulphonic acid (21.2 g, 0.22 mol)was added to the reaction drop-wise and the resulting white suspensionwas evaporated in vacuo. The white residue was quenched with water andextracted into chloroform (3×100 ml). The combined organic extracts werewashed with water (2×100 ml), dried over MgS O₄, and evaporated in vacuoto yield (3-oxo-1,3-dihydro-isobenzofuran-1-yl)phosphonic acid dimethylester as a white solid (32.0 g, 95%, 95% purity). This was then usedwithout further purification in the next stage.

To a mixture of (3-oxo-1,3-dihydro-isobenzofuran-1-yl)phosphonic aciddimethyl ester (35.0 g, 0.14 mol) in tetrahydrofuran (200 ml) and2-fluoro-5-formylbenzonitrile (20.9 g, 0.14 mol) in tetrahydrofuran (130ml) was added triethylamine (14 ml, 0.14 mol) drop-wise over 25 min,with the temperature kept below 15° C. The reaction mixture was warmedslowly to 20° C. over 1 hour and concentrated in vacuo. The whiteresidue was slurried in water (250 ml) for 30 minutes, filtered, washedwith water, hexane and ether, and dried to yield2-fluoro-5-(3-oxo-3H-isobenzofuran-1-ylidenemethyl)benzonitrile as a50:50 mixture of E and Z isomers (37.2 g, 96%); m/z [M+1]⁺ 266 (98%purity)

To a suspension of2-fluoro-5-(3-oxo-3H-isobenzofuran-1-ylidenemethyl)benzonitrile in water(200 ml) was added aqueous sodium hydroxide (26.1 g in 50 ml water)solution and the reaction mixture was heated under nitrogen to 90° C.for 30 minutes. The reaction mixture was partially cooled to 70° C., andhydrazine hydrate (100 ml) was added and stirred for 18 hours at 70° C.The reaction was cooled to room temperature and acidified with 2M HCl topH 4. The mixture was stirred for 10 min and filtered. The resultingsolid was washed with water, hexane, ether, ethyl acetate and dried toyield 2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid asa pale pink powder (30.0 g, 77%). m/z [M+1]⁺ 299 (96% purity), δ_(H) 4.4(2H, s), 7.2-7.3 (1H, m), 7.5-7.6 (1H, m), 7.8-8.0 (4H, m), 8.2-8.3 (1H,m), 12.6 (1H, s).

c.1-[3-(4-Oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoyl]piperidine-4-carboxylicacid (C)

3-(4-Oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (A)(7.0 g, 0.25mol), ethyl isonipecotate (5 ml, 0.32 mol),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) (12.3 g, 0.32 mol) and N,N,-diisopropylethylamine (10.0 ml, 0.55mol) were added to dimethylacetamide (40 ml) and stirred for 18 h. Water(100 ml) was added to the reaction mixture and the product was extractedinto dichloromethane (4×50 ml). The combined organic layers were washedwith water (3×100 ml), dried over MgSO₄, filtered and evaporated invacuo to yield an oil. To a solution of the oil in tetrahydrofuran (100ml) was added 10% aqueous sodium hydroxide solution (20 ml) and thereaction was stirred for 18 hours. The reaction was concentrated, washedwith ethyl acetate (2×30 ml) and acidified with 2M HCl to pH 2. Theaqueous layer was extracted with dichloromethane (2×100 ml), then theextracts were dried over MgSO₄, filtered and evaporated to yield1-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoyl]piperidine-4-carboxylicacid (C) as a yellow solid (7.0 g, 65%), m/z [M+1]⁺ 392 (96% purity),δ_(H) 1.3-1.8 (5H, m), 2.8-3.1 (4H, m), 4.4 (2H, s), 7.2-7.3 (1H, m),7.3-7.4 (1H, m), 7.7-8.0 (5H, m), 8.2-8.3 (1H, m), 12.6 (1H, s).

d.1-[2-Fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoyl]piperidine-4-carboxylicacid (D)

2-Fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (B)(3.1g, 0.14 mol), ethyl isonipecotate (1.7 ml, 0.11 mol),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) (5.1 g, 0.13 mol) and N,N,-diisopropylethylamine (10.0 ml, 0.55mol) were added to dimethylacetamide (15 ml) and stirred for 18 hours.Water (100 ml) was added to the reaction mixture and the product wasextracted into dichloromethane (4×50 ml). The combined organic layerswere, filtered, washed with water (3×100 ml), dried over MgSO₄, filteredand evaporated in vacuo to yield an orange oil. The oil was purified byflash chromatography (ethyl acetate) to yield1-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoyl]piperidine-4-carboxylicacid as the methyl ester (1.5 g, 33%, 96% purity). To a solution of themethyl ester in tetrahydrofuran:water (2:1, 40 ml) was added sodiumhydroxide (0.3 g, 0.075 mol) and the reaction was stirred for 18 h. Thereaction was concentrated, washed with ethyl acetate (2×20 ml) andacidified with 2M HCl to pH 2. The aqueous layer was extracted withdichloromethane (2×20 ml), and the combined extracts were dried overMgSO₄ and evaporated to yield1-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoyl]piperidine-4-carboxylicacid (D) as a yellow solid (0.6 g, 65%), m/z [M+1]⁺ 392 (96% purity)

Example 1 a. Synthesis of4-[3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (1)

3-(4-Oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (A)(5.0 g, 0.17mol), tert-butyl 1-piperazinecarboxylate (3.9 g, 0.21 mol),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) (8.6 g, 0.22 mol) and N,N,-diisopropylethylamine (6.7 ml, 0.38mol) were added to dimethylacetamide (40 ml) and stirred for 18 hours.Water (100 ml) was added and the reaction mixture was heated to 100° C.for 1 hour. The suspension was cooled to room temperature, filtered anddried to yield a white solid. The solid was dissolved in a solution of6M HCl and ethanol (2:1, 50 ml) and stirred for 1 hour. The reaction wasconcentrated, basified with ammonia to pH 9, and the product wasextracted into dichloromethane (2×50 ml). The combined organic layerswere washed with water (2×50 ml), dried over MgSO₄, and evaporated invacuo to yield 4-[3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one(1) as a yellow crystalline solid (4.0 g, 77%); m/z [M+1]⁺ 349 (97%purity), δ_(H) 2.6-3.8 (8H, m), 4.4 (2H, s), 7.2-7.5 (4H, m), 7.7-8.0(3H, m), 8.2-8.3 (1H, m), 12.6 (1H, s)

b. Synthesis of4-[4-Fluoro-3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (2)

The synthesis was carried out according to the method described in (a)above using 2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoicacid (B) to yield4-[4-fluoro-3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (2) asa white crystalline solid (4.8 g, 76%); m/z [M+1]⁺ 367 (97% purity),δ_(H) 2.6-3.8 (8H, m), 4.4 (2H, s), 7.2-7.5 (3H, m), 7.7-8.0 (3H, m),8.2-8.3 (1H, m), 12.6 (1H, s).

c. Synthesis of4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (3)

The synthesis was carried out according to the method described in (a)above using 3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (A)and tert-butyl 1-homopiperazine carboxylate to yield4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (3) as agrey crystalline solid (5.3 g, 97%); m/z [M+1]⁺ 363 (97% purity); δ_(H)2.6-3.8 (10H, m), 4.4 (2H, s), 7.2-7.5 (4H, m), 7.7-8.0 (3H, m), 8.2-8.3(1H, m), 12.6 (1H, s).

d. Synthesis of4-[3-([1,4]diazepane-1-carbonyl)-4-fluorobenzyl]-2H-phthalazin-1-one (4)

The synthesis was carried out according to the method described in (a)above using 2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoicacid (B) and tert-butyl 1-homopiperazinecarboxylate to yield4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (4) as ayellow crystalline solid (5.3 g, 68%); m/z [M+1]⁺ 381 (97% purity);δ_(H) 2.6-3.8 (10H, m), 4.4 (2H, s), 7.2-7.5 (3H, m), 7.7-8.0 (3H, m),8.2-8.3 (1H, m), 12.6 (1H, s).

Example 2 a.4-{3-[4-(6-Chlorobenzothiazol-2-yl)-1,4-diazepan-1-ylcarbonyl]benzyl}-1(2H)-phthalazinone

2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(150 mg, 0.47 mmol), diisopropylethylamine (102 mg, 0.8 mmol) and6-chloro-2-(1,4-diazepan-1-yl)-1,3-benzothiazole (115 mg, 0.43 mmol)were added sequentially at ambient temperature to a stirred solution of3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (A)(100 mg, 0.36mmol) in dry dimethylacetamide (1 ml), the mixture was stirred atambient temperature for 1 hour and allowed to stand at ambienttemperature for 16 hours, then it was added dropwise to stirred coldwater (10 ml). After 30 minutes, the resulting solid was collected byfiltration, washed with water (2×1 ml) and hexane (1 ml), dried in vacuoand purified using preparative HPLC to give the desired compound (5)(166mg) as a grey solid; HPLC purity 90%; HPLC Retention time 4.21 minutes;m/z (M+H)⁺ 530.

b. The Following Compounds were Synthesised in a Manner Analogous tothat Described in (a) Above, but Using Appropriate Alternative AmineStarting Materials

LC LC RT Purity Compound R (minutes) M + 1 (%)  6

4.18 508 90  7

3.22 551 90  8

4.13 508 90  9

3.95 483 90  10

3.79 465 90  11

3.76 406 90 219

2.80 407 90

LC Com- LC RT Purity pound R (minutes) M + 1 (%) 12 (Note 1)

3.56 494 100 13

3.71 451  90 14

4.39 538  90 15

3.66 498  90 16

4.33 533  90 Note 1: 12 did not require purification via preparativescale HPLC - the product from the reaction was essentially pure.

LC LC RT Purity Compound R (minutes) M + 1 (%) 17

4.64 526 90 18

3.99 482 90 19

4.00 452 90 20

4.15 466 90

Example 3 a.4-{3-[4-(4-fluorophenyl)piperazin-1-ylcarbonyl]benzyl}-1(2H)-phthalazinone(21)

2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(150 mg, 0.47 mmol), diisopropylethylamine (102 mg, 0.8 mmol) and1-(4-fluorophenyl)piperazine (65 mg, 0.47 mmol) were added sequentiallyat ambient temperature to a stirred solution of3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (A)(100 mg, 0.36mmol) in dry dimethylacetamide (1 ml), the mixture was stirred atambient temperature for 4 hours and allowed to stand at ambienttemperature for 16 hours, then it was added dropwise to stirred coldwater (10 ml). After 30 minutes, the resulting solid was collected byfiltration, washed with water (2×1 ml) and hexane (1 ml), dried in vacuoand purified using preparative HPLC to give4-{3-[4-(4-fluorophenyl)piperazin-1-ylcarbonyl]benzyl}-1(2H)-phthalazinone(21)(76 mg) as a cream solid; m/z (M+H)⁺ 443; HPLC Purity 90%; HPLCRetention time 4.00 minutes.

b. The Following Compounds were Synthesised in a Manner Analogous tothat Described in (a) Above, but Using Appropriate Alternative AmineStarting Materials

LC RT LC Purity Compound R (minutes) M + 1 (%)  22

4.00 470  90  23

4.26 486  90  24

3.18 504  85  25

3.78 473  90  26

4.46 583  90  27

4.96 509  90  28

3.73 511  90  29

3.78 553  90  30

3.71 459  90  31

3.94 546  90  32

3.84 485  90  33

4.37 552  90  34

3.77 485  90 220 (Note 2)

2.89 440 100 Note 2: 220 did not require purification via preparativescale HPLC - the product from the reaction was essentially pure.

Example 4

1-[3-(4-Oxo-3,4-dihydro-phthalazin-1-ylmethyl)-benzoyl]-piperidine-4-carboxylicacid (C) (0.24 mmol) was added to a solution of the appropriate amine(0.2 mmol) in dimethyl acetamide (2 ml).2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(0.3 mmol) and Hunigs base (0.4 mmol) were then added and the reactionwas stirred at room temperature for 16 hours. The reaction mixtures werethen purified by preparative HPLC.

The compounds synthesised are set out below.

LC RT LC Purity Compound R (minutes) M + 1 (%)  35

4.39 572 90  36

3.71 496 90  37

3.63 474 80  38

3.76 474 90  39

3.56 502 90  40

3.58 568 90  41

3.81 508 90  42

4.39 531 90  43

3.52 460 85  44

3.77 508 90  45

3.59 488 90  46

3.83 488 90  47

3.85 488 90  48

3.47 448 90  49

3.36 446 90  50

3.77 488 90  51

3.74 472 90  52

3.82 498 90  53

3.52 460 90  54

3.86 510 90  55

3.75 546 90  56

3.01 565 90  57

3.93 549 90 373

4.17 663 90 374

3.08 595 90 375

4.16 551 90 376

4.3  565 90 377

4.1  571 90 378

3.64 567 90 379

3.62 579 90 380

4.27 605 90 381

3.89 555 90 382

3.84 565 90 383

2.92 565 90 384

3.02 543 90

Example 5

The appropriate sulphonyl chloride (0.24 mmol) was added to a solutionof 4-[3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (1) (0.2mmol) in dichloromethane (2 ml). Hunigs base (0.4 mmol) was then addedand the reaction was stirred at room temperature for 16 hours. Thereaction mixtures were then purified by preparative HPLC.

The compounds synthesised are set out below.

LC RT LC Purity Compound R (minutes) M + 1 (%) 58

3.85 504 90 59

3.44 442 90 60

4.09 558 90 61

3.93 525 90 62

3.73 543 90 63

4.38 532 90 64

3.76 509 90 65

3.82 470 90

Example 6

The appropriate acid chloride (0.24 mmol) was added to a solution of4-[3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (1) (0.2 mmol)in dichloromethane (2 ml). Hunigs base (0.4 mmol) was then added and thereaction was stirred at room temperature for 16 hours. The reactionmixtures were then purified by preparative HPLC.

The compounds synthesised are set out below.

LC RT LC Purity Compound R (minutes) M + 1 (%) 66

4.04 530 85 67

3.71 460 90 68

3.69 506 90 69

3.19 450 85 70

3.44 478 90 71

3.94 538 90 72

3.68 526 90 73

3.6 464 90 74

3.63 472 90 75

3.73 535 80 76

3.49 530 90 77

3.5 512 85 78

3.52 459 90 79

4.01 588 90 80

4.05 406 90 81

3.84 513 90 82

4.07 520 90 83

4.41 671 90 84

3.62 582 90 85

3.82 508 90 86

3.81 507 90 87

3.33 445 90 88

4.08 571 90 89

3.67 480 90 90

3.54 577 90 91

3.49 498 90 92

4.04 510 90 93

3.75 512 90 94

3.67 482 90 95

3.54 474 90 96

3.52 537 90 97

4.13 475 90 98

3.8 512 85 99

4.09 544 90 100

3.63 486 90 101

3.91 502 90 102

3.61 511 90 103

3.57 474 90 104

3.67 504 90 105

4.02 508 90 106

3.81 500 90 107

4.11 540 90 108

4.19 560 90 109

3.61 468 90 110

3.69 582 90 111

3.85 549 85 112

4.37 573 90 113

3.84 496 90 114

3.62 573 90 115

3.72 500 90 116

3.8 500 85 117

3.9 496 90 118

4.03 560 90 119

4.16 560 90 120

4.71 472 80 121

3.47 526 90 122

3.78 632 90 123

3.27 506 90 124

3.92 590 90 125

4.76 706 90 126

4.27 605 90 127

3.71 557 90 128

3.98 551 90 129

3.9 496 90 130

3.57 454 90 131

3.21 450 90 132

3.61 446 90 133

3.39 418 85 134

3.81 494 90 135

3.31 418 90 136

3.38 444 90 137

3.56 506 85 138

3.48 484 90 139

3.84 510 90 140

3.56 472 90 141

3.34 476 90 142

3.56 490 90 143

3.53 488 90 144

3.99 523 90 145

3.7 468 90 192

3.11 392 90 350

3.16 462 90 351

2.77 474 90

Example 7

The appropriate isocyanate (0.24 mmol) was added to a solution of4-[3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (1) (0.2 mmol)in dichloromethane (2 ml). The reaction was stirred at room temperaturefor 16 hours. The reaction mixtures were then purified by preparativeHPLC.

The compounds synthesised are set out below.

LC RT LC Purity Compound R (minutes) M + 1 (%) 146

3.34 435 85 147

3.76 483 90 148

3.49 449 90 149

3.57 487 90 150

4.03 537 90 151

3.77 519 85 152

3.72 487 90 153

3.57 505 90 154

4.08 537 80 155

3.68 497 85 156

4.03 553 90 157

3.93 497 90 158

3.62 494 90 159

3.68 497 90 160

3.73 475 90 161

3.9 513 90 162

4.11 511 90 163

3.58 483 90 164

3.71 517 90 165

3.34 435 85 166

3.71 497 90 167

3.56 513 90 168

4.04 552 90 169

4 547 90 170

3.54 507 90 171

3.42 497 90 172

3.95 552 90 173

3.79 541 85 174

3.66 529 90 175

3.92 527 85 176

3.62 527 90 177

4.28 569 90 178

3.81 610 90 352

3.73 555 90 353

3.79 541 90

Example 8

The appropriate isothiocyanate (0.24 mmol) was added to a solution of4-[3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (1) (0.2 mmol)in dichloromethane (2 ml). The reaction was stirred at room temperaturefor 16 hours. The reaction mixtures were then purified by preparativeHPLC.

The compounds synthesised are set out below.

LC RT LC Purity Compound R (minutes) M + 1 (%) 179

3.68 515 90 180

4.05 513 90 181

3.94 465 90 182

3.55 449 90 183

4.21 575 90 184

3.79 543 90 185

4.28 557 85 186

3.63 527 90 187

3.18 528 90 188

3.32 423 90 189

3.69 485 80 190

3.68 515 90 191

3.72 503 90 354

4.67 542 90 355

3.96 543 90

Example 9

3-(4-Oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (A) (0.24 mmol)was added to a solution of the appropriate amine (0.2 mmol) indimethylacetamide (2 ml).2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(0.3 mmol) and Hunigs base (0.4 mmol) were then added and the reactionwas stirred at room temperature for 16 hours. The reaction mixtures werethen purified by preparative HPLC.

The compounds synthesised are set out below.

LC RT LC Purity Compound R (minutes) M + 1 (%) 193

2.72 378 90 221

2.77 407 90

LC RT LC Purity Compound R (minutes) M + 1 (%) 194

2.96 427 90 195

4.08 444 90 196

3.9 456 95 197

3.83 450 95 198

2.98 432 90 199

4.17 440 90 200

2.9 449 90 201

4.31 460 90 202

3.63 468 90 203

3.78 456 90 204

3.08 378 90

LC RT LC Purity Compound R (minutes) M + 1 (%) 205

2.88 432 90 206

3.61 421 95 207

3.96 397 90 356

4.34 564 90 357

3.84 554 90

Example 10

2-Fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (B)(0.24 mmol) was added to a solution of the appropriate amine (0.2 mmol)in dimethylacetamide (2 ml).2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(0.3 mmol) and Hunigs base (0.4 mmol) were then added and the reactionwas stirred at room temperature for 16 hours. The reaction mixtures werethen purified by preparative HPLC.

The compounds synthesised are set out below.

LC RT LC Purity Compound R (minutes) M + 1 (%) 208

2.8 396 90 359

2.78 425 85

LC RT LC Purity Compound R (minutes) M + 1 (%) 209

3.9 456 95 210

3.97 467 90 211

2.84 426 90 212

3.46 368 90 222

5.07 461 90 223

4.46 511 90 224

2.96 499 90 225

3.55 445 85 226

4.19 457 90 227

4.34 471 90 228

2.88 466 90 229

4.23 477 90 230

4.03 473 90 231

3.07 500 90 232

3.92 473 90 233

4.55 471 90 234

4.32 477 90 235

3.94 443 90 236

4.03 461 90 237

2.69 411 90 238

2.76 438 90 239

2.77 494 90 240

2.91 444 90 358

4.59 472 90

Com- LC RT LC Purity pound R (minutes) M + 1 (%) 213

3.81 439 90 214

3.95 415 90

LC RT LC Purity Compound R (minutes) M + 1 (%) 360

4.08 472 90

Example 11

An appropriate aldehyde (0.2 mmol) and4-[3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (1)(0.24 mmol)were dissolved in dichloromethane (2 ml). Sodium triacetoxyborohydride(0.28 mmol) and glacial acetic acid (6.0 mmol) were then added andstirred at room temperature for 16 hours. The reaction mixtures werethen purified by preparative HPLC.

The compounds synthesised are set out below.

LC RT LC Purity Compound R (minutes) M + 1 (%) 215

2.89 406 90 216

2.91 406 90 217

3.16 493 90 218

3.09 479 90 361

2.98 419 90 362

2.74 377 90 363

3.04 419 90 364

2.82 391 85 365

2.84 403 90 366

2.96 419 90 367

3.15 445 90 368

3.04 419 90 369

2.75 391 85

Example 12

An appropriate aldehyde (0.2 mmol) and4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (4)(0.24mmol) were dissolved in dichloromethane (2 ml). Sodiumtriacetoxyborohydride (0.28 mmol) and glacial acetic acid (6.0 mmol)were then added and stirred at room temperature for 16 hours. Thereaction mixtures were then purified by preparative HPLC.

The compounds synthesised are set out below.

LC RT LC Purity Compound R (minutes) M + 1 (%) 241

2.9  437 90 242

3.05 451 90 243

2.84 409 90 244

3.12 465 90 245

3.16 451 90 246

2.86 423 90 247

2.89 435 90 248

3.04 451 90 249

3.23 477 90 250

3.09 451 90 370

2.80 423 90

Example 13

An appropriate aldehyde (0.2 mmol) and4-[4-fluoro-3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one(2)(0.24 mmol) were dissolved in dichloromethane (2 ml). Sodiumtriacetoxyborohydride (0.28 mmol) and glacial acetic acid (6.0 mmol)were then added and stirred at room temperature for 16 hours. Thereaction mixtures were then purified by preparative HPLC.

The compounds synthesised are set out below.

LC RT LC Purity Compound R (minutes) M + 1 (%) 251

2.97 423 90 252

3.06 437 90 253

2.8  395 90 254

3.11 451 90 255

3.09 437 90 256

2.87 409 90 257

2.89 421 90 258

3.01 437 85 259

3.14 463 90 260

3.08 437 90 261

2.83 458 90 262

3.04 464 90 263

2.79 409 90

Example 14

The appropriate acid chloride (0.24 mmol) was added to a solution of4-[4-fluoro-3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (2)(0.2 mmol) in dichloromethane (2 ml). Hunigs base (0.4 mmol) was thenadded and the reaction was stirred at room temperature for 16 hours. Thereaction mixtures were then purified by preparative HPLC.

The compounds synthesised are set out below.

Com- LC RT LC Purity pound R (minutes) M + 1 (%) 264

3.89 409 90 265

4.17 462 85 266

4.04 423 90 267

4.03 417 90 268

3.19 480 90 269

2.84 492 90 270

2.71 521 90 271

2.83 508 90 272

2.86 478 90 273

2.63 521 90

Example 15

An appropriate aldehyde (0.2 mmol) and4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (3)(0.24mmol) were dissolved in dichloromethane (2 ml). Sodiumtriacetoxyborohydride (0.28 mmol) and glacial acetic acid (6.0 mmol)were then added and stirred at room temperature for 16 hours. Thereaction mixtures were then purified by preparative HPLC.

The compounds synthesised are set out below.

LC Com- LC RT Purity pound R (minutes) M + 1 (%) 274

2.92 419 90 275

3.04 433 90 276

2.78 391 90 277

3.09 433 90 278

2.86 405 90 279

2.88 417 90 280

2.99 433 90 281

3.11 459 90 282

3.06 433 90 283

2.93 443 90 284

2.92 451 90 285

2.99 459 90 286

2.94 441 90 287

3.36 589 85 288

2.72 487 85 289

3.24 499 90 290

3.14 497 90 291

3.9  483 85 292

3.22 493 90 293

2.91 419 90 294

3.08 511 90 295

2.92 443 90 296

3.03 478 90 297

3.48 501 90 298

3.39 545 90 299

3.06 456 90 300

3.8  483 90 301

3.08 472 90 302

3.22 506 90 303

3.13 522 90 304

3.29 533 90 305

3.39 589 90 306

3.07 492 90 307

3.41 589 90 308

2.87 460 85 309

3.06 496 90 310

3.13 513 90 311

2.96 459 90 312

2.98 453 90 313

3.09 501 90 314

2.76 405 90

Example 16

The appropriate sulphonyl chloride (0.24 mmol) was added to a solutionof 4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (3) (0.2mmol) in dichloromethane (2 ml). Hunigs base (0.4 mmol) was then addedand the reaction was stirred at room temperature for 16 hours. Thereaction mixtures were then purified by preparative HPLC.

The compounds synthesised are set out below.

Com- LC RT LC Purity pound R (minutes) M + 1 (%) 315

4.15 571 90 316

4.36 579 90 317

3.68 483 90 371

3.71 523 90

Example 17

The appropriate acid chloride (0.24 mmol) was added to a solution of4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (3) indichloromethane (2 ml). Hunigs base (0.4 mmol) was then added and thereaction was stirred at room temperature for 16 hours. The reactionmixtures were then purified by preparative HPLC.

The compounds synthesised are set out below.

LC Com- LC RT Purity pound R (minutes) M + 1 (%) 318

3.84 533 90 319

3.23 458 90 320

3.63 532 90 321

3.61 525 90 322

3.63 495 90 323

3.62 513 90 324

3.16 415 90 325

3.52 459 90 326

3.95 489 90 327

3.89 549 90 328

4.00 557 90

Example 18

The appropriate isocyanate (0.24 mmol) was added to a solution of4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (3) (0.2mmol) in dichloromethane (2 ml). The reaction was stirred at roomtemperature for 16 hours. The reaction mixtures were then purified bypreparative HPLC.

The compounds synthesised are set out below.

Com- LC RT LC Purity pound R (minutes) M + 1 (%) 329

3.4  512 90 330

3.95 550 90 331

3.76 510 90 332

3.58 526 90 333

4.04 582 90

Example 19

The appropriate isothiocyanate (0.24 mmol) was added to a solution of4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (3) (0.2mmol) in dichloromethane (2 ml). The reaction was stirred at roomtemperature for 16 hours. The reaction mixtures were then purified bypreparative HPLC.

The compounds synthesised are set out below.

Com- LC RT LC Purity pound R (minutes) M + 1 (%) 334

3.86 512 90 335

3.75 516 90 336

3.66 556 90 337

3.57 540 90 338

4.17 569 90 339

3.56 540 90 340

3.1  541 85 341

3.84 498 90 342

3.64 556 90 343

3.53 528 90 344

3.92 556 90 345

4.09 566 90 346

3.64 516 90 347

3.57 528 90 348

3.78 512 90 349

3.62 512 90

Example 20

The appropriate acid chloride (0.24 mmol) was added to a solution of4-[3-([1,4]diazepane-1-carbonyl)-4-fluorobenzyl]-2H-phthalazin-1-one (4)in dichloromethane (2 ml). Hunigs base (0.4 mmol) was then added and thereaction was stirred at room temperature for 16 hours. The reactionmixtures were then purified by preparative HPLC.

The compounds synthesised are set out below.

LC RT LC Purity Compound R (minutes) M + 1 (%) 372

2.81 466 80 385

3.19 495 90

Example 21

1-[2-Fluoro-5-(4-Oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoyl]-piperidine-4-carboxylicacid (D) (0.24 mmol) was added to a solution of the appropriate amine(0.2 mmol) in dimethylacetamide (2 ml).2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(0.3 mmol) and Hunigs base (0.4 mmol) were then added and the reactionwas stirred at room temperature for 16 hours. The reaction mixtures werethen purified by preparative HPLC.

The compounds synthesised are set out below.

LC RT LC Purity Compound R (minutes) M + 1 (%) 386

3.21 480 90

Example 22

In order to assess the inhibitory action of the compounds, the followingassay was used to determine IC₅₀ values (Dillon, et al., JBS., 8(3),347-352 (2003)).

Mammalian PARP, isolated from Hela cell nuclear extract, was incubatedwith Z-buffer (25 mM Hepes (Sigma); 12.5 mM MgCl₂ (Sigma); 50 mM KCl(Sigma); 1 mM DTT (Sigma); 10% Glycerol (Sigma) 0.001% NP-40 (Sigma); pH7.4) in 96 well FlashPlates (TRADE MARK) (NEN, UK) and varyingconcentrations of said inhibitors added. All compounds were diluted inDMSO and gave final assay concentrations of between 10 and 0.01 μM, withthe DMSO being at a final concentration of 1% per well. The total assayvolume per well was 40 μl.

After 10 minutes incubation at 30□C the reactions were initiated by theaddition of a 10 μl reaction mixture, containing NAD (5 μM), ³H-NAD and30mer double stranded DNA-oligos. Designated positive and negativereaction wells were done in combination with compound wells (unknowns)in order to calculate % enzyme activities. The plates were then shakenfor 2 minutes and incubated at 30□C for 45 minutes.

Following the incubation, the reactions were quenched by the addition of50 μl 30% acetic acid to each well. The plates were then shaken for 1hour at room temperature.

The plates were transferred to a TopCount NXT (TRADE MARK) (Packard, UK)for scintillation counting. Values recorded are counts per minute (cpm)following a 30 second counting of each well.

The % enzyme activity for each compound is then calculated using thefollowing equation:

${\% \mspace{14mu} {Inhibition}} = {100 - \left( {100 \times \frac{\left( {{{cpm}\mspace{14mu} {of}\mspace{14mu} {unknowns}} - {{mean}\mspace{14mu} {negative}\mspace{14mu} {cpm}}} \right)}{\left( {{{mean}\mspace{14mu} {positive}\mspace{14mu} {cpm}} - {{mean}\mspace{14mu} {negative}\mspace{14mu} {cpm}}} \right)}} \right)}$

IC₅₀ values (the concentration at which 50% of the enzyme activity isinhibited) were calculated, which are determined over a range ofdifferent concentrations, normally from 10 μM down to 0.001 μM. SuchIC₅₀ values are used as comparative values to identify increasedcompound potencies.

All compounds tested had a IC₅₀ of less than 0.1 μM.

The following compounds have an IC₅₀ of less than 0.01 μM: 2-5, 9, 10,12-20, 24, 26-28, 30, 32-35, 38, 39, 42, 44-47, 49, 51, 58-68, 80-82,84, 86, 88-90, 94-97, 100-105, 113-116, 121-123, 126, 132, 136, 138-140,142-144, 147, 149, 151, 152, 156, 158, 159, 161-164, 166-175, 177-180,182-184, 186-190, 194, 199, 202, 205, 207, 208, 213, 221-223, 225,233-236, 239-274, 277, 279, 287, 288, 292, 293, 301-303, 306, 315, 316,318-323, 325, 327-336, 338-350, 352-357, 359, 361, 363, 365 and 367-370.

The following compounds, as well as those above, have an IC₅₀ of lessthan 0.02 μM: 1, 6-8, 11, 21-23, 25, 31, 36, 37, 40, 41, 43, 48, 52-54,56, 57, 69-79, 83, 87, 91-93, 98, 99, 106, 109, 110, 111, 117, 118, 120,124, 128-130, 133-135, 137, 141, 145, 146, 148, 150, 153-155, 157, 160,165, 176, 181, 185, 191, 192, 195, 196, 197, 201, 203, 204, 206, 211,212, 215-217, 219, 220, 224, 226, 227, 229-232, 237, 238, 275, 276, 278,281-286, 289-291, 294, 295, 297-299, 304, 305, 307-309, 311, 314, 317,324, 326, 337, 351, 358, 360, 362, 364, 366, 371 and 372.

The Potentiation Factor (PF₅₀) for compounds is calculated as a ratio ofthe IC₅₀ of control cell growth divided by the IC₅₀ of cell growth+PARPinhibitor. Growth inhibition curves for both control and compoundtreated cells are in the presence of the alkylating agent methylmethanesulfonate (MMS). The test compounds were used at a fixedconcentration of 0.2 micromolar. The concentrations of MMS were over arange from 0 to 10 μg/ml.

Cell growth was assessed using the sulforhodamine B (SRB) assay (Skehan,P., et al., (1990) New colorimetric cytotoxicity assay foranticancer-drug screening. J. Natl. Cancer Inst. 82, 1107-1112.). 2,000HeLa cells were seeded into each well of a flat-bottomed 96-wellmicrotiter plate in a volume of 100 □l and incubated for 6 hours at 37°C. Cells were either replaced with media alone or with media containingPARP inhibitor at a final concentration of 0.5, 1 or 5 μM. Cells wereallowed to grow for a further 1 hour before the addition of MMS at arange of concentrations (typically 0, 1, 2, 3, 5, 7 and 10 μg/ml) toeither untreated cells or PARP inhibitor treated cells. Cells treatedwith PARP inhibitor alone were used to assess the growth inhibition bythe PARP inhibitor.

Cells were left for a further 16 hours before replacing the media andallowing the cells to grow for a further 72 hours at 37° C. The mediawas then removed and the cells fixed with 100 μl of ice cold 10% (w/v)trichloroacetic acid. The plates were incubated at 4° C. for 20 minutesand then washed four times with water. Each well of cells was thenstained with 100 μl of 0.4% (w/v) SRB in 1% acetic acid for 20 minutesbefore washing four times with 1% acetic acid. Plates were then driedfor 2 hours at room temperature. The dye from the stained cells wassolubilized by the addition of 100 μl of 10 mM Tris Base into each well.Plates were gently shaken and left at room temperature for 30 minutesbefore measuring the optical density at 564 nM on a Microquantmicrotiter plate reader.

All the compounds tested had a PF₅₀ at 200 nM of at least 2.0.

Example 23

To assess the stand alone activity of a PARP inhibitor on Braca 1 and 2deficient cells the following protocols were used.

Small Molecule Inhibitors of PARP:

Compound (4) was dissolved in DMSO at 10 mM and stored at −20° C. in thedark.

Cell Lines

VC8 cells and the mouse Brca2 BAC complemented derivatives were asdescribed in M. Kraakman-van der Zwet, et al., Mol Cell Biol 22, 669-79(2002)). ES cells defective in Brca2 function have been describedpreviously (Tutt, et al., EMBO Rep 3, 255-60 (2002)). The constructionof ES cells defective in Brca1 will be described elsewhere but havepreviously been validated (Foray, et al., Embo J, 22, 2860-71 (2003)).

Clonogenic Assays

For measurement of cellular sensitivity to a PARP inhibitor (compound4), cell cultures in exponential growth were trypsinised and seeded atvarious densities in 6-well plates onto Mitomycin C inactivated mouseembryonic fibroblasts and where appropriate treated with the testcompound after 18 hours. For continuous exposure, cells were re-fedevery 4 days with fresh medium and inhibitor. After 10-14 days, cellswere washed with PBS, fixed in methanol and stained with crystal violet.Colonies containing greater than approximately 50 cells were counted.Experiments were performed at least three times in triplicate.

Results Reduction in the Viability of BRCA1 and BRCA2 Deficient Cells

Compound 4 was used to probe the sensitivity of cells deficient in Brca1or Brca2 to the inhibition of PARP activity. Clonogenic assays showedthat both Brca1 and Brca2 deficient cell lines were extremely sensitiveto compound 4 compared to otherwise isogenic cells (FIG. 1A, 1B). TheSF₅₀ (dosage at which 50% of cells survived) for Compound 4 was1.5×10⁻⁸M for cells deficient in Brca1, whilst the SF₅₀ for matched wildtype cells was 7×10⁻⁶M (FIG. 1A). This represents a factor of 467 foldenhanced sensitivity of Brca1 mutant cells compared to wild type cells.

The SF₅₀ for Compound 4 was 1.2×10-8M for cells deficient in Brca2whilst the SF₅₀ for matched wild type cells was 1.8×10⁻⁵M (FIG. 1B).This represents a factor of 1,500 fold enhanced sensitivity of Brca2mutant cells compared to wild type cells. Similar results were obtainedwith Chinese hamster ovary cells deficient in Brca2 (VC8) compared to aBrca2-complemented derivative (VC8-BAC)(FIG. 2). The SF₅₀ for Compound 4was 5×10⁻⁸M for the Brca2 deficient VC8 line whilst the SF₅₀ for matchedcontrol, VC8-BAC, was 3×10⁻⁵M (FIG. 2). This represents a factor of 600fold enhanced sensitivity of Brca2 mutant cells compared to wild typecells.

1. A compound of formula (III):

or isomers, salts, or solvates thereof in combination with temozolimide.2. A pharmaceutical composition comprising a combination of a compoundof formula

or isomers, salts, or solvates thereof and temozolimide.
 3. Apharmaceutical composition according to claim 2, in the form of anintravenous preparation.
 4. A pharmaceutical composition according toclaim 2, in a form suitable for oral administration.
 5. A method oftreatment of cancer comprising administering to a subject in need oftreatment a therapeutically effective amount of a compound of formula(III):

simultaneously or sequentially with temozolimide.
 6. A method accordingto claim 5, wherein said cancer is deficient in a HR dependent DNA DSBrepair pathway.
 7. A method according to claim 6, comprising the step ofidentifying the subject as having a cancer condition which is deficientin a HR dependent DNS DSB repair pathway.
 8. A method according to claim6, wherein said cancer comprises one or more cancer calls having areduced or abrogated ability to repair DNA DSB by HR relative to normalcells.
 9. A method according to claim 8, wherein said cancer cells havea BRCA1 or BRCA2 deficient phenotype.
 10. A method according to claim 9,wherein said cancer cells are deficient in BRCA1 or BRCA2.
 11. A methodaccording to claim 6, wherein said cancer is breast, ovarian, pancreasor prostate cancer.
 12. A method according to claim 6, wherein saidpatient is heterozygous for a mutation in a gene encoding a component ofthe HR dependent DNA DSB repair pathway.